Methods for detection of a target nucleic acid by forming a cleavage structure using an RNA polymerase

ABSTRACT

The invention relates to compositions and methods for generating a signal indicative of the presence of a target nucleic acid in a sample, where the compositions and methods include an RNA polymerase, a FEN nuclease, and a probe.

RELATED APPLICATIONS

This application is a continuation-in-part which claims priority under35 U.S.C. §120 to U.S. patent application Ser. No. 09/728,574 filed Nov.30, 2000, which is a continuation-in-part of U.S. patent applicationSer. No. 09/650,888 filed Aug. 30, 2000 (now U.S. Pat. No. 6,548,250),which is a continuation-in-part of U.S. patent application Ser. No.09/430,692 filed Oct. 29, 1999 (now U.S. Pat. No. 6,528,254), theentireties of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates in general to methods of detecting or measuring atarget nucleic acid.

BACKGROUND OF THE INVENTION

The fidelity of DNA replication, recombination, and repair is essentialfor maintaining genome stability, and these processes depend on 5′→3′exonuclease enzymes which are present in all organisms. For DNA repair,these enzymes are required for damaged fragment excision andrecombinational mismatch correction. For replication, these nucleasesare critical for the efficient processing of Okazaki fragments duringlagging strand DNA synthesis. In Escherichia coli, this latter activityis provided by DNA polymerase I (PolI); E. coli strains withinactivating mutations in the Poll 5′□03′ exonuclease domain are notviable due to an inability to process Okazaki fragments. Eukaryotic DNApolymerases, however, lack an intrinsic 5′→3′ exonuclease domain, andthis critical activity is provided by the multifunctional,structure-specific metallonuclease FEN-1 (five′ exonuclease-1 or flapendonuclease-1), which also acts as an endonuclease for 5′ DNA flaps(Reviewed in Hosfield et al., 1998a, Cell, 95:135).

Methods of detecting and/or measuring a nucleic acid wherein an enzymeproduces a labeled nucleic acid fragment are known in the art.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose a method of cleaving a target DNA molecule by incubating a 5′labeled target DNA with a DNA polymerase isolated from Thermus aquaticus(Taq polymerase) and a partially complementary oligonucleotide capableof hybridizing to sequences at the desired point of cleavage. Thepartially complementary oligonucleotide directs the Taq polymerase tothe target DNA through formation of a substrate structure containing aduplex with a 3′ extension opposite the desired site of cleavage whereinthe non-complementary region of the oligonucleotide provides a 3′ armand the unannealed 5′ region of the substrate molecule provides a 5′arm. The partially complementary oligonucleotide includes a 3′nucleotide extension capable of forming a short hairpin either whenunhybridized or when hybridized to a target sequence at the desiredpoint of cleavage. The release of labeled fragment is detected followingcleavage by Taq polymerase.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose the generation of mutant, thermostable DNA polymerases thathave very little or no detectable synthetic activity, and wild typethermostable nuclease activity. The mutant polymerases are said to beuseful because they lack 5′ to 3′ synthetic activity; thus syntheticactivity is an undesirable side reaction in combination with a DNAcleavage step in a detection assay.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose that wild type Taq polymerase or mutant Taq polymerases thatlack synthetic activity can release a labeled fragment by cleaving a 5′end labeled hairpin structure formed by heat denaturation followed bycooling, in the presence of a primer that binds to the 3′ arm of thehairpin structure. Further, U.S. Pat. Nos. 5,843,669, 5,719,028,5,837,450, 5,846,717 and 5,888,780 teach that the mutant Taq polymeraseslacking synthetic activity can also cleave this hairpin structure in theabsence of a primer that binds to the 3′ arm of the hairpin structure.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose that cleavage of this hairpin structure in the presence ofa primer that binds to the 3′ arm of the hairpin structure by mutant Taqpolymerases lacking synthetic activity yields a single species oflabeled cleaved product, while wild type Taq polymerase producesmultiple cleavage products and converts the hairpin structure to adouble stranded form in the presence of dNTPs, due to the high level ofsynthetic activity of the wild type Taq enzyme.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose that mutant Taq polymerases exhibiting reduced syntheticactivity, but not wild type Taq polymerase, can release a single labeledfragment by cleaving a linear nucleic acid substrate comprising a 5′ endlabeled target nucleic acid and a complementary oligonucleotide whereinthe complementary oligonucleotide hybridizes to a portion of the targetnucleic acid such that 5′ and 3′ regions of the target nucleic acid arenot annealed to the oligonucleotide and remain single stranded.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose a method of cleaving a labeled nucleic acid substrate atnaturally occurring areas of secondary structure. According to thismethod, biotin labeled DNA substrates are prepared by PCR, mixed withwild type Taq polymerase or CleavaseBN (a mutant Taq polymerase withreduced synthetic activity and wild type 5′ to 3′ nuclease activity),incubated at 95° C. for 5 seconds to denature the substrate and thenquickly cooled to 65° C. to allow the DNA to assume its unique secondarystructure by allowing the formation of intra-strand hydrogen bondsbetween the complementary bases. The reaction mixture is incubated at65° C. to allow cleavage to occur and biotinylated cleavage products aredetected.

Methods of detecting and/or measuring a nucleic acid wherein a FEN-1enzyme is used to generate a labeled nucleic acid fragment are known inthe art.

U.S. Pat. No. 5,843,669 discloses a method of detecting polymorphisms bycleavase fragment length polymorphism analysis using a thermostableFEN-1 nuclease in the presence or absence of a mutant Taq polymeraseexhibiting reduced synthetic activity. According to this method, doublestranded Hepatitis C virus (HCV) DNA fragments are labeled by using 5′end labeled primers (labeled with TMR fluorescent dye) in a PCRreaction. The TMR labeled PCR products are denatured by heating to 950 Cand cooled to 550 C to generate a cleavage structure. U.S. Pat. No.5,843,669 discloses that a cleavage structure comprises a region of asingle stranded nucleic acid substrate containing secondary structure.Cleavage is carried out in the presence of CleavaseBN nuclease, FEN-1nuclease derived from the archaebacteria Methanococcus jannaschii orboth enzymes. Labeled reaction products are visualized by gelelectrophoresis followed by fluoroimaging. U.S. Pat. No. 5,843,669discloses that CleavaseBN nuclease and Methanococcus jannaschii FEN-1nuclease produce cleavage patterns that are easily distinguished fromeach other, and that the cleavage patterns from a reaction containingboth enzymes include elements of the patterns produced by cleavage witheach individual enzyme but are not merely a composite of the cleavagepatterns produced by each individual enzyme. This indicates that some ofthe fragments that are not cleaved by one enzyme (and which appear as aband in that enzyme's pattern) can be cleaved by a second enzyme in thesame reaction mixture.

Lyamichev et al. disclose a method for detecting DNAs whereinoverlapping pairs of oligonucleotide probes that are partiallycomplementary to a region of target DNA are mixed with the target DNA toform a 5′ flap region, and wherein cleavage of the labeled downstreamprobe by a thermostable FEN-1 nuclease produces a labeled cleavageproduct. Lyamichev et al. also disclose reaction conditions whereinmultiple copies of the downstream oligonucleotide probe can be cleavedfor a single target sequence in the absence of temperature cycling, soas to amplify the cleavage signal and allow quantitative detection oftarget DNA at sub-attomole levels (Lyamichev et al., 1999, Nat.Biotechnol., 17:292).

The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat.Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is anin vitro method for the enzymatic synthesis of specific DNA sequences,using two oligonucleotide primers that hybridize to opposite strands andflank the region of interest in the target DNA. A repetitive series ofreaction steps involving template denaturation, primer annealing and theextension of the annealed primers by DNA polymerase results in theexponential accumulation of a specific fragment whose termini aredefined by the 5′ ends of the primers. PCR is reported to be capable ofproducing a selective enrichment of a specific DNA sequence by a factorof 10⁹. The PCR method is also described in Saiki et al., 1985, Science,230:1350.

While the PCR technique is an extremely powerful method for amplifyingnucleic acid sequences, the detection of the amplified material requiresadditional manipulation and subsequent handling of the PCR products todetermine whether the target DNA is present. It is desirable to decreasethe number of subsequent handling steps currently required for thedetection of amplified material. An assay system, wherein a signal isgenerated while the target sequence is amplified, requires fewerhandling steps for the detection of amplified material, as compared to aPCR method that does not generate a signal during the amplificationstep.

U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose a PCR based assay forreleasing labeled probe comprising generating a signal during theamplification step of a PCR reaction in the presence of a nucleic acidto be amplified, Taq polymerase that has 5′ to 3′ exonuclease activityand a 5′, 3′ or 5′ and 3′ end-labeled probe comprising a regioncomplementary to the amplified region and an additionalnon-complementary 5′ tail region. U.S. Pat. Nos. 5,210,015 and 5,487,972disclose further that this PCR based assay can liberate the 5′ labeledend of a hybridized probe when the Taq polymerase is positioned near thelabeled probe by an upstream probe in a polymerization independentmanner, e.g. in the absence of dNTPs.

SUMMARY OF THE INVENTION

The invention provides a method of generating a signal indicative of thepresence of a target nucleic acid in a sample, which includes the stepsof forming a cleavage structure by incubating a sample containing atarget nucleic acid with a probe and an RNA polymerase, and cleaving thecleavage structure with a nuclease to release a nucleic acid fragmentand thus generate a signal.

In a first aspect, the invention is directed to a composition forgenerating a signal indicative of the presence of a target nucleic acidsequence in a sample. The composition includes an RNA polymerase, a FENnuclease, a primer and a probe.

In another aspect, the invention is directed to a composition forperforming a non-polymerase chain reaction process for simultaneouslyforming a cleavage structure, amplifying a target nucleic acid andcleaving the cleavage structure. The composition includes an RNApolymerase, a FEN nuclease and a probe.

In still another aspect, the invention is directed to a composition forgenerating a signal indicative of the presence of a target nucleic acidsequence in a sample. The composition includes an RNA polymerase, a FENnuclease and a probe that is complementary to the target nucleic acid,wherein the probe forms a cleavage structure when the 5′ end of theprobe is displaced by an RNA synthesized by the RNA polymerase.

In one embodiment, the FEN nuclease and RNA polymerase are thermostable.The FEN nuclease can be derived from Archaeglobus fulgidus,Methanococcus jannaschii, Pyrococcus furiosus, Taq, Tfl and Bca. In oneembodiment, the RNA polymerase is a DNA dependent RNA polymerase. In analternative embodiment, the RNA polymerase is an RNA dependent RNApolymerase. The RNA polymerase may initiate the synthesis of an RNA froman RNA promoter region or a primer. Suitable RNA polymerases include:T7-RNA polymerase, SP6-RNA polymerase, T3 RNA polymerase or NS5B RNApolymerase from hepatitis C virus (HCV).

The probe includes a 5′ region and a 3′ region. The 3′ region of theprobe is at least partially complementary to the target. The 5′ regionmay be complementary to the target or may be non-complementary to thetarget and therefore form a 5′ flap. In some embodiments, the probe islabeled with one or more detectable labels. For example, the probe mayinclude a pair of interactive signal generating labeled moietieseffectively positioned to quench the generation of a detectable signal.The labeled moieties are separated by a site susceptible to FEN nucleasecleavage, thereby allowing the nuclease activity of the FEN nuclease toseparate the first interactive signal generating labeled moiety from thesecond interactive signal generating labeled moiety by cleaving at thesite susceptible to FEN nuclease. When the interactive signal generatinglabeled moieties are separated a detectable signal is produced. Suitableinteractive signal generating moieties include a quencher and fluorescerpair.

In some embodiments, the compositions may further include one or moreupstream oligonucleotide primers that hybridizes upstream of the probe.In some embodiments, the primer is extended by the RNA polymerase. Inother embodiments, the primer encodes an RNA promoter region which isincorporated into the target.

In yet another aspect, the invention provides kits for any of the abovecompositions. The kits include a FEN nuclease, an RNA polymerase, aprobe and a suitable buffer. In one embodiment, the FEN nuclease and RNApolymerase are thermostable. The FEN nuclease can be derived fromArchaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus,Taq, Tfl and Bca. In another embodiment, the RNA polymerase is a DNAdependent RNA polymerase. In an alternative embodiment, the RNApolymerase is an RNA dependent RNA polymerase. Suitable RNA polymerasesinclude: T7-RNA polymerase, SP6-RNA polymerase, T3 RNA polymerase orNS5B RNA polymerase from hepatitis C virus (HCV).

In another aspect, the invention provides a method for generating asignal indicative of the presence of a target nucleic acid sequence in asample. The method is performed by forming a cleavage structure byincubating a sample having a target nucleic acid sequence, with an RNApolymerase and cleaving the cleavage structure with a FEN nuclease togenerate a signal. The signal is indicative of the presence of a targetnucleic acid sequence in the sample. Cleavage of the cleavage structurecan release nucleic acid fragments which are detected and/or measured.Alternatively, the cleaved probe is directly detected and/or measured.

In one embodiment, the RNA polymerase and FEN nuclease are thermostable.The RNA polymerase may be an RNA dependent or DNA dependent RNApolymerase. In another embodiment, RNA polymerase synthesizes an RNAthat is complementary to the target. The RNA polymerase may initiate thesynthesis of the complementary RNA strand via a promoter region orprimer. Suitable RNA polymerases include: T7-RNA polymerase, SP6-RNApolymerase, T3 RNA polymerase or NS5B RNA polymerase from hepatitis Cvirus (HCV).

In one embodiment, the FEN nuclease and RNA polymerase are thermostable.The FEN nuclease can be derived from Archaeglobus fulgidus,Methanococcus jannaschii, Pyrococcus furiosus, Taq, Tfl and Bca.

In another aspect, the invention provides a method for detecting atarget nucleic acid sequence in a sample by mixing a probe, a targetnucleic acid having a promoter region, a FEN nuclease and an RNApolymerase. The mixture is subjected to conditions which are permissivefor the steps of (i) binding of the RNA polymerase to the promoterregion in the target nucleic acid sequence and hybridization of theprobe to the target, (ii) synthesizing an RNA polymerase extensionproduct, (iii) forming a cleavage structure, and (iv) cleaving thecleavage structure with the FEN nuclease thereby generating a detectablesignal. The signal is detected and/or measured. The promoter regiontarget may be endogenously present in the target or it may by createdprior to or during the detection reaction. For example, a primer may beused which encodes an RNA promoter region which is incorporated into thetarget prior to the detection reaction. Such techniques are known in theart (e.g., 3SR, NASBA) and disclosed herein.

In yet another aspect, the invention provides a method for detecting atarget nucleic acid sequence in a sample by mixing a downstream probe, atarget nucleic acid, an upstream primer, a FEN nuclease and an RNApolymerase. The mixture is subjected to conditions which are permissivefor the steps of (i) annealing of the upstream primer and downstreamprobe to the target, (ii) synthesizing an RNA polymerase extensionproduct from the primer, (iii) forming a cleavage structure, and (iv)and cleaving the cleavage structure with the FEN nuclease therebygenerating a detectable signal. The signal is detected and/or measured.Such methods can be used with primer dependent RNA polymerases.

The RNA polymerase may be an RNA dependent or DNA dependent RNApolymerase. Suitable RNA polymerases include both DNA dependent and RNAdependent RNA polymerases, e.g., T7-RNA polymerase, SP6-RNA polymerase,T3 RNA polymerase or NS5B RNA polymerase from (HCV).

The FEN nuclease may be thermostable. Suitable FEN nucleases are derivedfrom: Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcusfuriosus, Taq, Tfl and Bca.

In one embodiment, the step of detecting and/or measuring a signalcomprises detecting a change in fluorescence between an interactive pairof labels. In yet another embodiment, the step of detecting and/ormeasuring a signal comprises detecting or measuring the release oflabeled fragments.

In another embodiment, the probe has at least one labeled moiety capableof providing a signal. In a further embodiment, the probe has a pair ofinteractive signal generating labeled moieties. The labeled moieties areeffectively positioned to quench the generation of a detectable signalwhen the probe is uncleaved. The labeled moieties are separated by asite susceptible to FEN nuclease cleavage, thereby allowing the nucleaseactivity of the FEN nuclease to separate the first interactive signalgenerating labeled moiety from the second interactive signal generatinglabeled moiety by cleaving at the site susceptible to FEN nuclease. Theseparation of the first and second interactive signal generating labeledmoieties generates a detectable signal. Suitable interactive pairs oflabels include a quencher moiety and a fluorescer moiety.

In another embodiment of either of the last two aspects, the RNApolymerase polymerizes nucleotides complementary to a length of thetarget sufficient to form a cleavage structure so that the polymerizedcomplementary RNA is adjacent at its 3′ end to the probe. In yet anotherembodiment of either of the last two aspects, the RNA polymerasedisplaces at least a portion of the probe sufficient to form a cleavagestructure.

As used herein, the term “probe” refers to a labeled oligonucleotidewhich forms a duplex structure with a sequence in the target nucleicacid, due to complementarity of at least one sequence in the probe witha sequence in the target region. The probe, preferably, does not containa sequence complementary to sequence(s) used in a primer. The probecomprises a region or regions that are complementary to a target nucleicacid (e.g., target nucleic acid binding sequences) (for example C inFIG. 4). In some embodiments, a “probe” according to the invention has asecondary structure that changes upon binding of the probe to the targetnucleic acid and further comprises a binding moiety. A “probe” accordingto the invention binds to a target nucleic acid to form a cleavagestructure that can be cleaved by a nuclease, wherein cleaving isperformed at a cleaving temperature, and wherein the secondary structureof the probe when not bound to the target nucleic acid is, preferably,stable at or below the cleaving temperature. A probe according to theinvention cannot be cleaved to generate a signal by a “nuclease”, asdefined herein, prior to binding to a target nucleic acid. In oneembodiment of the invention, a probe may comprise a region that cannotbind or is not complementary to a target nucleic acid. In anotherembodiment of the invention, a probe does not have a secondary structurewhen bound to a target nucleic acid.

As used herein, “secondary structure” refers to a three-dimensionalconformation (for example a hairpin, a stem-loop structure, an internalloop, a bulge loop, a branched structure or a pseudoknot, FIGS. 1 and 3;multiple stem loop structures, cloverleaf type structures or any threedimensional structure. As used herein, “secondary structure” includestertiary, quaternary etc. . . . structure. A probe comprising such athree-dimensional structure binds to a target nucleic acid to form acleavage structure that can be cleaved by a nuclease at a cleavingtemperature. The three dimensional structure of the probe when not boundto the target nucleic acid is, preferably, stable at or below thecleaving temperature. “Secondary structure” as used herein, can mean asequence comprising a first single-stranded sequence of bases (referredto herein as a “complementary nucleic acid sequence” (for example b inFIG. 4)) followed by a second complementary sequence either in the samemolecule (for example b′ in FIG. 4), or in a second molecule comprisingthe probe, folds back on itself to generate an antiparallel duplexstructure, wherein the single-stranded sequence and the complementarysequence (that is, the complementary nucleic acid sequences) anneal bythe formation of hydrogen bonds. Oligonucleotide probes, as used in thepresent invention include oligonucleotides comprising secondarystructure, including, but not limited to molecular beacons, safety pins(FIG. 9), scorpions (FIG. 10), and sunrise/amplifluor probes (FIG. 11),the details and structures of which are described below and in thecorresponding figures.

As used herein, first and second “complementary” nucleic acid sequencesare complementary to each other and can anneal by the formation ofhydrogen bonds between the complementary bases.

A secondary structure also refers to the conformation of a nucleic acidmolecule comprising an affinity pair, defined herein, wherein theaffinity pair reversibly associates as a result of attractive forcesthat exist between the pair of moieties comprising the affinity pair. Asused herein, secondary structure prevents the binding moiety on theprobe from binding to a capture element, and a change in secondarystructure upon binding of the probe to the target nucleic acid andsubsequent cleavage of the bound probe permits the binding moiety to becaptured by the capture element.

A “probe” according to the invention can be unimolecular. As usedherein, a “unimolecular” probe comprises a single molecule that binds toa target nucleic acid to form a cleavage structure that can be cleavedby a nuclease, wherein cleaving is performed at a cleaving temperature,and wherein the secondary structure of the “unimolecular” probe when notbound to the target nucleic acid is, preferably, stable at or below thecleaving temperature. Unimolecular probes useful according to theinvention include but are not limited to beacon probes, probescomprising a hairpin, stem-loop, internal loop, bulge loop or pseudoknotstructure, scorpion probes and sunrise/amplifluor probes.

A “probe” according to the invention can be more than one molecule(e.g., bi-molecular or multi-molecular). At least one of the moleculescomprising a bi-molecular or multi-molecular probe binds to a targetnucleic acid to form a cleavage structure that can be cleaved by anuclease, wherein cleaving is performed at a cleaving temperature, andwherein the secondary structure of the molecule of the probe when notbound to the target nucleic acid is, preferably, stable at or below thecleaving temperature. The molecules comprising the multimolecular probeassociate with each other via intermolecular bonds (e.g., hydrogen bondsor covalent bonds). For example, a heterologous loop (see FIG. 1), or acloverleaf structure wherein one or more loops of the cloverleafstructure comprises a distinct molecule, and wherein the molecules thatassociate to form the cloverleaf structure associate via intermolecularbonding (e.g., hydrogen bonding or covalent bonding), are examples ofmultimolecular probes useful according to the invention.

As used herein, a “molecule” refers to a polynucleotide, and includes apolynucleotide further comprising an attached member or members of anaffinity pair.

A “probe” or a “molecule” comprising a probe is 5-10,000 nucleotides inlength, ideally from 6-5000, 7-1000, 8-500, 9-250, 10-100 and 17-40nucleotides in length, although probes or a molecule comprising a probeof different lengths are useful.

A “probe” according to the invention has a target nucleic acid bindingsequence that is from 5 to 10,000 nucleotides, and preferably from 10 toabout 140 nucleotides. A “probe” according to the invention comprises atleast first and second complementary nucleic acid sequences or regionsthat are 3-250, preferably 4-150, and more preferably 5-110 and mostpreferably 6-50 nucleotides long. The first and second complementarynucleic acid sequences may have the same length or may be of differentlengths. The invention provides for a probe wherein the first and secondcomplementary nucleic acid sequences are both located upstream (5′) ofthe target nucleic acid binding site. Alternatively, the first andsecond complementary nucleic acid sequences can both be locateddownstream (3′) of the target nucleic acid binding site. In anotherembodiment, the invention provides for a probe wherein the firstcomplementary nucleic acid sequence is upstream (5′) of the targetnucleic acid binding site and the second complementary nucleic acidsequence is downstream (3′) of the target nucleic acid binding site. Inanother embodiment, the invention provides for a probe wherein thesecond complementary nucleic acid sequence is upstream (5′) of thetarget nucleic acid binding site and the first complementary nucleicacid sequence is downstream (3′) of the target nucleic acid bindingsite. The actual length will be chosen with reference to the targetnucleic acid binding sequence such that the secondary structure of theprobe is, preferably, stable when the probe is not bound to the targetnucleic acid at the temperature at which cleavage of a cleavagestructure comprising the probe bound to a target nucleic acid isperformed. As the target nucleic acid binding sequence increases in sizeup to 500 nucleotides, the length of the complementary nucleic acidsequences may increase up to 15-125 nucleotides. For a target nucleicacid binding sequence greater than 100 nucleotides, the length of thecomplementary nucleic acid sequences need not be increased further. Ifthe probe is also an allele-discriminating probe, the lengths of thecomplementary nucleic acid sequences are more restricted, as isdiscussed below.

As used herein, the “target nucleic acid binding sequence” refers to theregion of the probe that binds specifically to the target nucleic acid.

A “hairpin structure” or a “stem” refers to a double-helical regionformed by base pairing between adjacent, inverted, complementarysequences in a single strand of RNA or DNA.

A “stem-loop” structure refers to a hairpin structure, furthercomprising a loop of unpaired bases at one end.

As used herein, a probe with “stable” secondary structure when not boundto a target nucleic acid, refers to a secondary structure wherein 50% ormore (e.g., 50%, 55%, 75% or 100%) of the base pairs that constitute theprobe are not dissociated under conditions which permit hybridization ofthe probe to the target nucleic acid, but in the absence of the targetnucleic acid.

“Stability” of a nucleic acid duplex is determined by the meltingtemperature, or “T_(m)”. The T_(m) of a particular nucleic acid duplexunder specified conditions (e.g., salt concentration and/or the presenceor absence of organic solvents) is the temperature at which half (50%)of the base pairs of the duplex molecule have disassociated (that is,are not hybridized to each other in a base-pair).

The “stability” of the secondary structure of a probe when not bound tothe target nucleic acid is defined in a melting temperature assay, in afluorescence resonance energy transfer (FRET) assay or in a fluorescencequenching assay, (the details or which are described in a sectionentitled, “Determining the Stability or the Secondary Structure of aProbe”).

In some embodiments, a probe useful in the invention preferably willhave secondary structure that is “stable”, when not bound to a target,at or below the temperature of the cleavage reaction. Thus, thetemperature at which nuclease cleavage of a probe/target nucleic acidhybrid is performed according to the invention, must be lower than theTm of the secondary structure. The secondary structure of the probe is“stable” in a melting temperature assay at a temperature that is at orbelow the temperature of the cleavage reaction (i.e., at which cleavageis performed) if the level of light absorbance at the temperature at orbelow the temperature of the cleavage reaction is less than (i.e., atleast 5% less than, preferably 20% less than and most preferably 25%less than etc. . . . ) than the level of light absorbance at atemperature that is equal to or greater than the Tm of the probe.

According to the method of the invention, the stability of a secondarystructure can be measured by a FRET assay or a fluorescence quenchingassay (described in the section entitled, “Determining the Stability ofthe Secondary Structure of a Probe”). As used herein, a fluorescencequenching assay can include a FRET assay. A probe according to theinvention is labeled with an appropriate pair of interactive labels(e.g., a FRET pair (for example as described in the section entitled,“Determining the Stability of the Secondary Structure of the Probe”,below) that can interact over a distance of, for example 2 nucleotides,or a non-FRET-pair, (e.g., tetramethylrhodamine and DABCYL) that caninteract over a distance of, for example, 20 nucleotides. For example, aprobe according to the invention may be labeled with a fluorophore and aquencher and fluorescence is then measured, in the absence of a targetnucleic acid, at different temperatures. The Tm is the temperature atwhich the level of fluorescence is 50% of the maximal level offluorescence observed for a particular probe, see FIG. 12 e. The Tm fora particular probe wherein the nucleic acid sequence of the probe isknown, can be predicted according to methods known in the art. Thus,fluorescence is measured over a range of temperatures, e.g., wherein thelower temperature limit of the range is at least 50° Celsius below, andthe upper temperature limit of the range is at least 50° Celsius abovethe Tm or predicted Tm, for a probe according to the invention.

A secondary structure is herein defined as “stable” in a FRET assay at atemperature that is at or below the cleaving temperature if the level orwavelength of fluorescence is increased or decreased (e.g., at least 5%less than, preferably 20% less than and more preferably 25% less than,etc. . . . ) as compared with the level or wavelength of FRET that isobserved at the Tm of the probe (see FIGS. 12 e and f). For example, anincrease or a decrease in FRET can occur in a FRET assay according tothe invention. In another embodiment, a shift in wavelength, whichresults in an increase in the new, shifted wavelength or, a decrease inthe new shifted wavelength, can occur in a FRET assay according to theinvention.

A “change” in a secondary structure, according to the invention can bemeasured in a fluorescence quenching assay wherein a probe according tothe invention comprises a fluorophore and a quencher that are positionedsuch that in the absence of a target nucleic acid, and at temperaturesbelow the Tm of the probe there is quenching of the fluorescence (asdescribed above). As used herein, a “change” in secondary structure thatoccurs when a probe according to the invention binds to a target nucleicacid, refers to an increase in fluorescence in such an assay, such thatthe level of fluorescence after binding of the probe to the targetnucleic acid at a temperature below the Tm of the probe, is greater than(e.g., at least 5%, preferably 5-20% and most preferably 25% or more)the level of fluorescence observed in the absence of a target nucleicacid (see FIG. 12 g).

A secondary structure, according to the invention, can be detected bysubjecting a probe comprising a fluorophore and a quencher to afluorescence quenching assay (as described above). A probe that exhibitsa change in fluorescence that correlates with a change in temperature,see FIG. 12 e (e.g., fluorescence increases as the temperature of theFRET reaction is increased) may be capable of forming a secondarystructure.

As used herein, a “cleaving temperature” that is useful according to theinvention is a temperature that is less than (at least 1° C. andpreferably 10° C.) the T_(m) of a probe having a secondary structure.The “cleaving temperature” is initially selected to be possible andpreferably optimal for the particular nuclease being employed in thecleavage reaction.

Preferably the 3′ terminus of the probe will be “blocked” to prohibitincorporation of the probe into a primer extension product if an activepolymerase is used in the reaction. “Blocking” can be achieved by usingnon-complementary bases or by adding a chemical moiety such as biotin ora phosphate group to the 3′ hydroxl of the last nucleotide, which may,depending upon the selected moiety, serve a dual purpose by also actingas a label for subsequent detection or capture of the nucleic acidattached to the label. Blocking can also be achieved by removing the3′-OH or by using a nucleotide that lacks a 3′-OH such asdideoxynucleotide.

The term probe encompasses an allele-discriminating probe. As usedherein, an “allele-discriminating” probe preferentially hybridizes toperfectly complementary target nucleic acids and discriminates againstsequences that vary by at least one nucleotide. A nucleic acid sequencewhich differs by at least one nucleotide, as compared to a targetnucleic acid, hereafter referred to as a “target-like nucleic acidsequence”, is thus not a target nucleic acid for anallele-discriminating probe according to the invention.Allele-discriminating probes do not hybridize sufficiently to atarget-like nucleic acid sequence that contains one or more nucleotidemismatches as compared to the target nucleic acid complementarysequence, at a particular temperature or within a range of temperaturesdetermined by experimental optimization to permit an allelediscriminating probe to discriminate between a target and a target-likesequence with at least a single nucleotide difference, and thus do notundergo a change in secondary structure upon binding to a target-likenucleic acid sequence in the presence of only a target-like nucleic acidsequence, and under conditions that would support hybridization of theallele discriminating probe to a target nucleic acid.

In one embodiment, an “allele-discriminating probe” according to theinvention refers to a probe that hybridizes to a target-like nucleicacid sequence that varies by at least one nucleotide from the targetnucleic acid, wherein the variant nucleotide(s) is/are not located inthe allele-discriminating site. According to this embodiment of theinvention, “an allele-discriminating probe” cannot bind to a target-likenucleic acid sequence that also varies by at least one nucleotide in theallele-discriminating site, at a particular temperature or within arange of temperatures determined by experimental optimization to permitan allele discriminating probe to discriminate between a target and atarget-like sequence with at least a single nucleotide difference.Single nucleotide differences only affect the percentage of a probe thatis bound to a target or target-like nucleic acid sequence. For example,the invention provides for a perfectly matched probe, wherein as much as100% of the target or is in a probe-target complex (e.g., is bound byprobe), in the presence of excess probe. The invention also provides forprobes comprising at least a single base mismatch wherein at least 1-5%and preferably 5-10% of the target-like sequence is bound by the probeunder the same conditions used to form a complex comprising a targetsequence and a perfectly matched probe.

As used herein, “allele-discriminating site” refers to a region of atarget nucleic acid that is different (i.e., by at least one nucleotide)from the corresponding region in all possible alleles comprising thetarget nucleic acid.

Allele-discriminating probes useful according to the invention alsoinclude probes that bind less effectively to a target-like sequence, ascompared to a target sequence. The effectiveness of binding of a probeto a target sequence or a target-like sequence can be measured in a FRETassay, performed at a temperature that is below (at least 1° C. andpreferably 110° C. or more) the Tm of the secondary structure of theprobe, in the presence of a target-like sequence or a target sequence.The change in the level of fluorescence in the presence or absence of atarget sequence compared to the change in the level of fluorescence inthe presence or absence of a target-like sequence, provides an effectivemeasure of the effectiveness of binding of a probe to a target ortarget-like sequence.

In a method according to the invention, a probe that binds lesseffectively to a target-like sequence as compared to a target sequencewould undergo a smaller (e.g., preferably 25-50%, more preferably 50-75%and most preferably 75-90% of the value of the change in fluorescenceupon binding to a target nucleic acid) change in secondary structure, asdetermined by measuring fluorescence in a FRET or fluorescence quenchingassay as described herein, upon hybridization to a target-like sequenceas compared to a target nucleic acid. In a method according to theinvention, a probe that binds less effectively to a target-like sequenceas compared to a target sequence would generate a signal that isindicative of the presence of a target-like nucleic acid sequence in asample. However, the intensity of the signal would be altered (e.g.,preferably 25-50%, more preferably 50-75% and most preferably 75-90%less than or more than the value of the change in fluorescence uponbinding to a target nucleic acid) the intensity of a signal generated inthe presence of a target sequence, as described hereinabove for asmaller change.

A “signal that is indicative of the presence of a target nucleic acid”or a “target-like nucleic acid sequence” refers to a signal that isequal to a signal generated from 1 molecule to 10²⁰ molecules, morepreferably about 100 molecules to 10¹⁷ molecules and most preferablyabout 1000 molecules to 10¹⁴ molecules of a target nucleic acid or atarget-like nucleic acid sequence.

As used herein, a “binding moiety” refers to a region of a probe (forexample ab in FIG. 4) that is released upon cleavage of the probe by anuclease and binds specifically to a capture element as a result ofattractive forces that exist between the binding moiety and the captureelement, and wherein specific binding between the binding moiety and thecapture element only occurs when the secondary structure of the probehas “changed”, as defined herein. “Binds specifically” means viahydrogen bonding with a complementary nucleic acid or via an interactionbetween for example, the binding moiety and a binding protein capable ofbinding specifically to the nucleic acid sequence of the binding moiety.A “binding moiety” does not interfere with the ability of a probe tobind to a target nucleic acid. A binding moiety is incapable of bindingto a capture element when the probe is in its native secondarystructural conformation and that, upon binding to a target or templatenucleic acid, the secondary structure changes in a way that allows thebinding moiety to bind to the capture element, preferably after cleavageby a cleavage agent.

In one embodiment, the region of a probe that is cleaved to form abinding moiety cannot hybridize to a target nucleic acid. The region ofa “binding moiety” that is not a “complementary nucleic acid sequence”,as defined herein, (e.g., A in FIG. 4), is from 1-60 nucleotides,preferably from 1-25 nucleotides and most preferably from 1-10nucleotides in length. Methods of detecting specific binding between abinding moiety or a binding moiety, as defined herein, and a captureelement, as defined herein, are well known in the art and are describedhereinbelow.

In one embodiment of the invention, a probe further comprises a“reporter”.

As used herein, a “reporter” refers to a “label”, defined hereinbelowand/or a “tag” defined hereinbelow.

As used herein, “label” or “labeled moiety capable of providing asignal” refers to any atom or molecule which can be used to provide adetectable (preferably quantifiable) signal, and which can beoperatively linked to a nucleic acid. Labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, massspectrometry, binding affinity, hybridization radiofrequency,nanocrystals and the like. A labeled probe according to the methods ofthe invention is labeled at the 5′ end, the 3′ end or internally. Thelabel can be “direct”, i.e. a dye, or “indirect”. i.e. biotin, digoxin,alkaline phosphatase (AP), horse radish peroxidase (HRP) etc. . . . Fordetection of “indirect labels” it is necessary to add additionalcomponents such as labeled antibodies, or enzyme substrates to visualizethe, captured, released, labeled nucleic acid fragment. In oneembodiment of the invention, a label cannot provide a detectable signalunless the secondary structure has “changed”, as defined herein, (forexample, such that the binding moiety is accessible).

A “binding moiety” also refers to a “tag”. As used herein, a “tag”refers to a moiety that is operatively linked to the 5′ end of a probe(for example R in FIG. 1) and specifically binds to a capture element asa result of attractive forces that exist between the tag and the captureelement, and wherein specific binding between the tag and the captureelement only occurs when the secondary structure of the probe haschanged (for example, such that the tag is accessible to a captureelement). “Specifically binds” as it refers to a “tag” and a captureelement means via covalent or hydrogen bonding or electrostaticattraction or via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, or a nucleic acidbinding protein and a nucleic acid binding site. A tag does notinterfere with the ability of a probe to anneal to a target nucleicacid. Tags include but are not limited to biotin, streptavidin, avidin,an antibody, an antigen, a hapten, a protein, or a chemically reactivemoiety. A “tag” as defined herein can bind to a “capture element” asdefined herein. According to the invention, a “tag” and a “captureelement” function as a binding pair. For example, in one embodiment, ifa tag is biotin, the corresponding capture element is avidin.Alternatively, in another embodiment, if a tag is an antibody, thecorresponding capture element is an antigen.

The invention contemplates a “probe” comprising a binding moiety, a“probe” comprising a “tag”, as defined herein, and a “probe” comprisingboth a binding moiety that is a region of a probe that is released uponcleavage of the probe by a nuclease (for example a nucleic acid sequencethat binds to a capture element), and a “tag”.

As used herein, a “capture element” refers to a substance that isattached to a solid substrate for example by chemical crosslinking orcovalent binding, wherein the substance specifically binds to (e.g., viahydrogen bonding or via an interaction between, a nucleic acid bindingprotein and a nucleic acid binding site or between complementary nucleicacids) a binding moiety as a result of attractive forces that existbetween the binding moiety and the capture element, and wherein specificbinding between the binding moiety and the capture element only occurswhen the secondary structure of the probe comprising the binding moietyhas “changed”, as defined herein. Capture elements include but are notlimited to a nucleic acid binding protein or a nucleotide sequence.

As used herein, a “capture element” also refers to a substance that isattached to a solid substrate for example by chemical crosslinking orcovalent binding, wherein the substance specifically binds to (e.g. viacovalent or hydrogen bonding or electrostatic attraction via aninteraction between, for example a protein and a ligand, an antibody andan antigen, protein subunits, a nucleic acid binding protein and anucleic acid binding site or between complementary nucleic acids) a tagas a result of attractive forces that exist between the tag and thecapture element, and wherein specific binding between the tag and thecapture element only occurs when the secondary structure of the probecomprising the tag has “changed”, as defined herein. Capture elementsinclude but are not limited to biotin, avidin, streptavidin, anantibody, an antigen, a hapten, a protein, or a chemically reactivemoiety. A “tag” as defined herein can bind to a “capture element” asdefined herein. According to the invention, a “tag” and a “captureelement” function as a binding pair. For example, in one embodiment, ifa capture element is biotin, the corresponding tag is avidin.Alternatively, in another embodiment, if a capture element is anantibody, the corresponding tag is an antigen.

As used herein, “solid support” means a surface to which a molecule(e.g. a capture element) can be irreversibly bound, including but notlimited to membranes, sepharose beads, magnetic beads, tissue cultureplates, silica based matrices, membrane based matrices, beads comprisingsurfaces including but not limited to styrene, latex or silica basedmaterials and other polymers for example cellulose acetate, teflon,polyvinylidene difluoride, nylon, nitrocellulose, polyester, carbonate,polysulphone, metals, zeolites, paper, alumina, glass, polypropyle,polyvinyl chloride, polyvinylidene chloride, polytetrafluorethylene,polyethylene, polyamides, plastic, filter paper, dextran, germanium,silicon, (poly)tetrafluorethylene, gallium arsenide, gallium phosphide,silicon oxide, silicon nitrate and combinations thereof. Methods ofattaching a capture element as defined herein are well known in the artand are defined hereinbelow. Additional solid supports are alsodiscussed hereinbelow.

As used herein, “affinity pair” refers to a pair of moieties (forexample complementary nucleic acid sequences, protein-ligand,antibody-antigen, protein subunits, and nucleic acid bindingproteins-binding sites) that can reversibly associate as a result ofattractive forces that exist between the moieties. An “affinity pair”includes the combination of a binding moiety and the correspondingcapture element and the combination of a tag and the correspondingcapture element.

In embodiments wherein the affinity pair comprises complementary nucleicacid regions that reversibly interact with one another, the lengths ofthe target nucleic acid binding sequences, and the nucleic acidsequences comprising the affinity pair, are chosen for the properthermodynamic functioning of the probe under the conditions of theprojected hybridization assay. Persons skilled in hybridization assayswill understand that pertinent conditions include probe, target andsolute concentrations, detection temperature, the presence ofdenaturants and volume excluders, and other hybridization-influencingfactors. The length of a target nucleic acid binding sequence can rangefrom 7 to about 10,000 nucleotides, preferably from 8-5000, 9-500, 9-250and most preferably, 10 to 140 nucleotides. If the probe is also anallele-discriminating probe, the length is more restricted, as isdiscussed below (this sentence has jumped in logic from a bindingmoiety:capture element concept to a probe:target concept).

In embodiments wherein the affinity pair comprises complementary nucleicacid regions that reversibly interact with one another, and cannothybridize or are not complementary to a target nucleic acid, thecomplementary nucleic acid region sequences of the affinity pair shouldbe of sufficient length that under the conditions of the assay and atthe detection temperature, when the probe is not bound to a target, thestructure of the probe is such that the binding moiety of the probe willnot bind to the capture element, e.g., the complementary nucleic acidsequences are associated. Depending upon the assay conditions used,complementary nucleic acid sequences of 3-25 nucleotide lengths canperform this function. An intermediate range of 4-15, and morepreferably 5-11, nucleotides is often appropriate. The actual lengthwill be chosen with reference to the target nucleic acid bindingsequence such that the secondary structure of the probe is stable whennot bound to the target nucleic acid at the temperature at whichcleavage of a cleavage structure comprising the probe bound to a targetnucleic acid is performed. As the target nucleic acid binding sequenceincreases in size up to 100 nucleotides, the length of the complementarynucleic acid sequences may increase up to 15-25 nucleotides. For atarget nucleic acid binding sequence greater than 100 nucleotides, thelength of the complementary nucleic acid sequences need not be increasedfurther. If the probe is also an allele-discriminating probe, thelengths of the complementary nucleic acid sequences are more restricted,as is discussed below.

Allele-discriminating probes that do not hybridize sufficiently to atarget-like nucleic acid sequence that contains one or more nucleotidemismatches as compared to the target nucleic acid complementarysequence, must be designed such that, under the assay conditions used,reduction or elimination of secondary structure in the probe andhybridization with a target nucleic acid will occur efficiently onlywhen the target nucleic acid complementary sequence finds a perfectlycomplementary target sequence under certain reaction conditions. Certainreaction conditions may include, for example, a particular temperatureor a range of temperatures determined by experimental optimization topermit an allele discriminating probe to discriminate between a targetand a target-like-sequence with at least a single nucleotide difference.

In one embodiment, an “allele-discriminating probe” according to theinvention refers to a probe that hybridizes to a target-like nucleicacid sequence that varies by at least one nucleotide from the targetnucleic acid, wherein the variant nucleotide(s) is/are not located inthe allele-discriminating site. According to this embodiment of theinvention, “an allele-discriminating probe” cannot bind efficiently to atarget-like nucleic acid sequence that also varies by at least onenucleotide in the allele-discriminating site under certain reactionconditions. Certain reaction conditions may include, for example, aparticular temperature or a range of temperatures determined byexperimental optimization to permit an allele discriminating probe todiscriminate between a target and a target-like sequence with at least asingle nucleotide difference.

In one embodiment of the invention, an allele discriminating probeaccording to the invention preferably comprises a target nucleic acidbinding sequence from 6 to 50 and preferably from 7 to 25 nucleotides,and complementary nucleic acid sequences from 3 to 8 nucleotides. Theguanosine-cytidine content of the secondary structure and probe-targethybrids, salt, and assay temperature should all be considered, forexample magnesium salts have a strong stabilizing effect that isparticularly important to consider when designing short,allele-discriminating probes.

If an allele-discriminating probe is to have a target nucleic acidbinding sequence of about 50 nucleotides long, the sequence should bedesigned such that a single nucleotide mismatch to be discriminatedagainst occurs at or near the middle of the target nucleic acidcomplementary sequence. For example, probes comprising a sequence thatis 21 nucleotides long should preferably be designed so that themismatch occurs opposite one of the 14 most centrally locatednucleotides of the target nucleic acid complementary sequence and mostpreferably opposite one of the 7 most centrally located nucleotides.Designing a probe so that the mismatch to be discriminated againstoccurs in or near the middle of the target nucleic acid bindingsequence/target-like nucleic acid binding sequence is believed toimprove the performance of an allele-discriminating probe.

As used herein a “nuclease” or a “cleavage agent” refers to an enzymethat is specific for, that is, cleaves a cleavage structure according tothe invention and is not specific for, that is, does not substantiallycleave either a probe or a primer that is not hybridized to a targetnucleic acid, or a target nucleic acid that is not hybridized to a probeor a primer. The term “nuclease” includes an enzyme that possesses 5′endonucleolytic activity for example a DNA polymerase, e.g. DNApolymerase I from E. coli, and DNA polymerase from Thermus aquaticus(Taq), Thermus thermophilus (Tth), and Thermus flavus (Tfl). The termnuclease also embodies FEN nucleases. The term “FEN nuclease”encompasses an enzyme that possesses 5′ exonuclease and/or anendonuclease activity. The term “FEN nuclease” also embodies a 5′flap-specific nuclease. A nuclease or cleavage agent according to theinvention includes but is not limited to a FEN nuclease enzyme derivedfrom Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcusfuriosus, human, mouse or Xenopus laevis. A nuclease according to theinvention also includes Saccharomyces cerevisiae RAD27, andSchizosaccharomyces pombe RAD2, Pol I DNA polymerase associated 5′ to 3′exonuclease domain, (e.g. E. coli, Thermus aquaticus (Taq), Thermusflavus (Tfl), Bacillus caldotenax (Bca), Streptococcus pneumoniae) andphage functional homologs of FEN including but not limited to T5 5′ to3′ exonuclease, T7 gene 6 exonuclease and T3 gene 6 exonuclease.Preferably, only the 5′ to 3′ exonuclease domains of Taq, Tfl and BcaFEN nuclease are used. The term “nuclease” does not include RNAse H.

As used herein, “captured” as it refers to capture of a binding moietyby a capture element or capture of a tag by a capture element, meansspecifically bound by hydrogen bonding, covalent bonding, or via aninteraction between, for example a protein and a ligand, an antibody andan antigen, protein subunits, a nucleic acid binding protein and anucleic acid binding site, or between complementary nucleic acids,wherein one member of the interacting pair is attached to a solidsupport. Under conditions of stable capture, binding results in theformation of a heterodimer with a dissociation constant (K_(D)) of atleast about 1×10³ M⁻¹, usually at least 1×10⁴ M⁻¹, typically at least1×10⁵ M⁻¹, preferably at least 1×10⁶ M⁻¹ to 1×10⁷ M⁻¹ or more, undersuitable conditions. Methods of performing binding reactions between acapture element, as defined herein, and a binding moiety or tag, asdefined herein, are well-known in the art and are described hereinbelow. Methods of attaching a capture element according to the inventionto a solid support, as defined herein, are well known in the art and aredefined hereinbelow.

As used herein, “wild type” refers to a gene or gene product which hasthe characteristics of (i.e., either has the sequence of or encodes, forthe gene, or possesses the sequence or activity of, for an enzyme) thatgene or gene product when isolated from a naturally occurring source.

A “5′ flap-specific nuclease” (also referred to herein as a“flap-specific nuclease”) according to the invention is an endonucleasewhich can remove a single stranded flap that protrudes as a 5′ singlestrand. In one embodiment of the invention, a flap-specific nucleaseaccording to the invention can also cleave a pseudo-Y structure. Asubstrate of a flap-specific nuclease according to the invention,comprises a target nucleic acid and an oligonucleotide probe, as definedherein, that comprises a region or regions that are complementary to thetarget nucleic acid. In another embodiment, a substrate of aflap-specific nuclease according to the invention comprises a targetnucleic acid, an upstream oligonucleotide that is complementary to thetarget nucleic acid and a downstream probe, according to the invention,that comprises a region or regions that are complementary to the targetnucleic acid. In one embodiment, the upstream oligonucleotide and thedownstream probe hybridize to non-overlapping regions of the targetnucleic acid. In another embodiment the upstream oligonucleotide and thedownstream probe hybridize to adjacent regions of the target nucleicacid.

As used herein, “adjacent” refers to separated by less than 20nucleotides, e.g., 15 nucleotides, 10 nucleotides, 5 nucleotides, or 0nucleotides.

A substrate of a flap-specific nuclease according to the invention, alsocomprises a target nucleic acid, a second nucleic acid, a portion ofwhich specifically hybridizes with a target nucleic acid, and a primerextension product from a third nucleic acid that specifically hybridizeswith a target nucleic acid.

As used herein, a “cleavage structure” refers to a polynucleotidestructure (for example as illustrated in FIG. 1) comprising at least aduplex nucleic acid having a single stranded region comprising a flap, aloop, a single-stranded bubble, a D-loop, a nick or a gap. A cleavagestructure according to the invention thus includes a polynucleotidestructure comprising a flap strand of a branched nucleic acid wherein a5′ single-stranded polynucleotide flap extends from a position near itsjunction to the double stranded portion of the structure and preferablythe flap is labeled with a detectable label. A flap of a cleavagestructure according to the invention is preferably about 1-10,000nucleotides, more preferably about 5-25 nucleotides and most preferablyabout 10-20 nucleotides and is preferably cleaved at a position locatedat the phosphate positioned at the “elbow” of the branched structure orat any of one to ten phosphates located proximal and/or distal from theelbow of the flap strand. As used herein, “elbow” refers to thephosphate bond between the first single stranded nucleotide of the 5′flap and the first double stranded (e.g., hybridized to the targetnucleic acid) nucleotide. In one embodiment, a flap of a cleavagestructure cannot hybridize to a target nucleic acid.

A cleavage structure according to one embodiment of the inventionpreferably comprises a target nucleic acid, and also may include anoligonucleotide probe according to the invention, that specificallyhybridizes with the target nucleic acid via a region or regions that arecomplementary to the target nucleic acid, and a flap extending from thehybridizing oligonucleotide probe. In another embodiment of theinvention, a cleavage structure comprises a target nucleic acid (forexample B in FIG. 4), an upstream oligonucleotide that is complementaryto the target sequence (for example A in FIG. 4), and a downstreamoligonucleotide probe according to the invention and comprising a regionor regions, that are complementary to the target sequence (for example Cin FIG. 4). In one embodiment, the upstream oligonucleotide and thedownstream probe hybridize to non-overlapping regions of the targetnucleic acid. In another embodiment, the upstream oligonucleotide andthe downstream probe hybridize to adjacent regions of the target nucleicacid.

A cleavage structure according to the invention may be a polynucleotidestructure comprising a flap extending from the downstreamoligonucleotide probe of the invention, wherein the flap is formed byextension of the upstream oligonucleotide by the synthetic activity of anucleic acid polymerase, and subsequent, partial, displacement of the 5′end of the downstream oligonucleotide. In such a cleavage structure, thedownstream oligonucleotide may be blocked at the 3′ terminus to preventextension of the 3′ end of the downstream oligonucleotide.

A cleavage structure according to one embodiment of the invention may beformed by hybridizing a target nucleic acid with an oligonucleotideprobe wherein the oligonucleotide probe has a secondary structure thatchanges upon binding of the probe to the target nucleic acid, andfurther comprises a binding moiety and a complementary region thatanneals to the target nucleic acid, and a non-complementary region thatdoes not anneal to the target nucleic acid and forms a 5′ flap.

A cleavage structure also may be a pseudo-Y structure wherein apseudoY-structure is formed if the strand upstream of a flap (referredto herein as a flap adjacent strand or primer strand) is not present,and double stranded DNA substrates containing a gap or nick. A “cleavagestructure”, as used herein, does not include a double stranded nucleicacid structure with only a 3′ single-stranded flap. As used herein, a“cleavage structure” comprises ribonucleotides or deoxyribonucleotidesand thus can be RNA or DNA.

A cleavage structure according to the invention may be an overlappingflap wherein the 3′ end of an upstream oligonucleotide capable ofhybridizing to a target nucleic acid (for example A in FIG. 4) isidentical to 1 base pair of the downstream oligonucleotide probe of theinvention (for example C in FIG. 4) that is annealed to a target nucleicacid and wherein the overlap is directly downstream of the point ofextension of the single stranded flap.

A cleavage structure according to one embodiment of the invention isformed by the steps of 1. incubating a) an upstream 3′ end, preferablyan oligonucleotide primer, b) an oligonucleotide probe located not morethan 10,000 nucleotides downstream of the upstream primer and comprisingat least one detectable label c) an appropriate target nucleic acidwherein the target sequence is at least partially complementary to boththe upstream primer and downstream probe and d) a suitable buffer, underconditions that allow the nucleic acid sequence to hybridize to theoligonucleotide primers, and, in one embodiment of the invention, 2.extending the 3′ end of the upstream oligonucleotide primer by thesynthetic activity of a polymerase, e.g., RNA polymerase, such that thenewly synthesized 3′ end of the upstream oligonucleotide primer becomesadjacent to and/or displaces at least a portion of (i.e., at least 1-10nucleotides of) the 5′ end of the downstream oligonucleotide probe.According to the method of the invention, buffers and extensiontemperatures are favorable for strand displacement by a particularnucleic acid polymerase according to the invention. Preferably, thedownstream oligonucleotide is blocked at the 3′ terminus to preventextension of the 3′ end of the downstream oligonucleotide.

In another embodiment of the invention, a cleavage structure accordingto the invention can be prepared by incubating a target nucleic acidwith an oligonucleotide probe having at least one detectable label, andfurther comprising a non-complementary 5′ region that does not anneal tothe target nucleic acid and forms a 5′ flap, and a complementary 3′region that anneals to the target nucleic acid.

In another embodiment of the invention, a cleavage structure accordingto the invention can be prepared by incubating a target nucleic acidwith a downstream oligonucleotide probe having a secondary structurethat changes upon binding of the probe to the target nucleic acid, andfurther comprising a binding moiety and a non-complementary 5′ regionthat does not anneal to the target nucleic acid and forms a 5′ flap anda complementary 3′ region that anneals to the target nucleic acid, andan upstream oligonucleotide primer. In one embodiment, the upstreamoligonucleotide and the downstream probe hybridize to non-overlappingregions of the target nucleic acid. In another embodiment, the upstreamoligonucleotide and the downstream probe hybridize to adjacent regionsof the target nucleic acid.

In another embodiment of the invention, a cleavage structure is formedby the steps of 1. incubating a) an oligonucleotide probe located notmore than 10,000 nucleotides downstream of a promoter region andcomprising at least one detectable label b) an appropriate targetnucleic acid wherein the target sequence comprises a promoter region andis at least partially complementary to downstream probe and c) asuitable buffer, under conditions that allow the nucleic acid sequenceto hybridize to the oligonucleotide probe and allow the promoter regionto bind the RNA polymerase, and 2. synthesizing an RNA by the syntheticactivity of an RNA polymerase such that the newly synthesized 3′ end ofthe synthesized RNA becomes adjacent to and/or displaces at least aportion of (i.e., at least 1-10 nucleotides of) the 5′ end of thedownstream oligonucleotide probe. According to the method of theinvention, buffers and extension temperatures are favorable for stranddisplacement by a particular RNA polymerase according to the invention.Preferably, the downstream oligonucleotide is blocked at the 3′ terminusto prevent extension of the 3′ end of the downstream oligonucleotide.

In a preferred embodiment of the invention a cleavage structure islabeled. A labeled cleavage structure according to one embodiment of theinvention is formed by the steps of 1. incubating a) an upstreamextendable 3′ end, for example, an oligonucleotide primer, b) a labeledprobe having a secondary structure that changes upon binding of theprobe to the target nucleic acid, and further comprising a bindingmoiety, preferably located not more than 10,000 and more preferablylocated not more than 500 nucleotides downstream of the upstream primerand c) an appropriate target nucleic acid wherein the target sequence iscomplementary to both the primer and the labeled probe and d) a suitablebuffer, under conditions that allow the nucleic acid sequence tohybridize to the primers, and, in one embodiment of the invention, 2.extending the 3′ end of the upstream primer by the synthetic activity ofa polymerase such that the newly synthesized 3′ end of the upstreamprimer partially displaces the 5′ end of the downstream probe. Accordingto the method of the invention, buffers and extension temperatures arefavorable for strand displacement by a particular nucleic acidpolymerase according to the invention. Preferably, the downstreamoligonucleotide is blocked at the 3′ terminus to prevent extension ofthe 3′ end of the downstream oligonucleotide. In one embodiment, theupstream primer and the downstream probe hybridize to non-overlappingregions of the target nucleic acid.

In another embodiment, a cleavage structure according to the inventioncan be prepared by incubating a target nucleic acid with a probe havinga secondary structure that changes upon binding of the probe to thetarget nucleic acid, and further comprising a binding moiety and anon-complementary, labeled, 5′ region that does not anneal to the targetnucleic acid and forms a 5′ flap, and a complementary 3′ region thatanneals to the target nucleic acid. In another embodiment, a cleavagestructure according to the invention can be prepared by incubating atarget nucleic acid with a downstream probe having a secondary structurethat changes upon binding of the probe to the target nucleic acid, andfurther comprising a binding moiety and a non-complementary, labeled, 5′region that does not anneal to the target nucleic acid and forms a 5′flap and a complementary 3′ region that anneals to the target nucleicacid, and an upstream oligonucleotide primer. In one embodiment, theupstream oligonucleotide and the downstream probe hybridize tonon-overlapping regions of the target nucleic acid. In anotherembodiment, the upstream oligonucleotide and the downstream probehybridize to adjacent regions of the target nucleic acid.

As used herein, “generating a signal” refers to detecting and ormeasuring a released nucleic acid fragment that is released from thecleavage structure as an indication of the presence of a target nucleicacid in a sample. In one embodiment, generating a signal also refers todetecting or measuring a released nucleic acid fragment that is capturedby binding of a binding moiety to a capture element on a solid support,as an indication of the presence of a target nucleic acid in a sample.

As used herein, “sample” refers to any substance containing or presumedto contain a nucleic acid of interest (a target nucleic acid) or whichis itself a nucleic acid containing or presumed to contain a targetnucleic acid of interest. The term “sample” thus includes a sample ofnucleic acid (genomic DNA, cDNA, RNA), cell, organism, tissue, fluid, orsubstance including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, synovial fluid, urine, tears, stool, externalsecretions of the skin, respiratory, intestinal and genitourinarytracts, saliva, blood cells, tumors, organs, tissue, samples of in vitrocell culture constituents, natural isolates (such as drinking water,seawater, solid materials), microbial specimens, and objects orspecimens that have been “marked” with nucleic acid tracer molecules.

As used herein, “target nucleic acid” or “template nucleic acidsequence” refers to a region of a nucleic acid that is to be eitherreplicated, amplified, and/or detected. In one embodiment, the “targetnucleic acid” or “template nucleic acid sequence” resides between twoprimer sequences used for amplification.

As used herein, “nucleic acid polymerase” refers to an enzyme thatcatalyzes the polymerization of nucleoside triphosphates. Generally, theenzyme will initiate synthesis at the 3′-end of the primer annealed tothe target sequence, and will proceed in the 5′-direction along thetemplate, and if possessing a 5′ to 3′ nuclease activity, hydrolyzingintervening, annealed probe to release both labeled and unlabeled probefragments, until synthesis terminates. Known DNA polymerases include,for example, E. coli DNA polymerase I, T7 DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNApolymerase, Thermococcus litoralis DNA polymerase, Thermus aquaticus(Taq) DNA polymerase and Pyrococcus furiosus (Pfu) DNA polymerase. Theterm “nucleic acid polymerase” also encompasses RNA polymerases.

As used herein, the term “RNA polymerase” refers to an enzyme thatcatalyzes the polymerization of an RNA molecule. RNA polymeraseencompasses DNA dependant as well as RNA dependent RNA polymerases.Suitable RNA polymerases for use in the invention include bacteriophageT7, T3 and SP6 RNA polymerases, E. coli RNA polymerase holoenzyme, E.coli RNA polymerase core enzyme, and human RNA polymerase I, II, III,human mitochondrial RNA polymerase and NS5B RNA polymerase from (HCV).

As used herein, “5′ to 3′ exonuclease activity” or “5′→3′ exonucleaseactivity” refers to that activity of a template-specific nucleic acidpolymerase e.g. a ′→3′ exonuclease activity traditionally associatedwith some DNA polymerases whereby mononucleotides or oligonucleotidesare removed from the 5′ end of a polynucleotide in a sequential manner,(i.e., E. coli DNA polymerase I has this activity whereas the Klenow(Klenow et al., 1970, Proc. Natl. Acad. Sci., USA, 65:168) fragment doesnot, (Klenow et al., 1971, Eur. J. Biochem., 22:371)), orpolynucleotides are removed from the 5′ end by an endonucleolyticactivity that may be inherently present in a 5′ to 3′ exonucleaseactivity.

As used herein, the phrase “substantially lacks 5′ to 3′ exonucleaseactivity” or “substantially lacks 5′→3′ exonuclease activity” meanshaving less than 10%, 5%, 1%, 0.5%, or 0.1% of the activity of a wildtype enzyme. The phrase “lacking 5′ to 3′ exonuclease activity” or“lacking 5′→3′ exonuclease activity” means having undetectable 5′ to 3′exonuclease activity or having less than about 1%, 0.5%, or 0.1% of the5′ to 3′ exonuclease activity of a wild type enzyme.

To detect structure-specific endonucleolytic activity, a DNA templateconsisting of a flap structure, wherein the downstream flapoligonucleotide is radiolabeled at the 5′ end is employed. The reactionis carried out with DNA polymerase in the presence of dNTPs (to extendthe upstream primer). Radiolabeled cleavage products are visualized bygel electrophoresis (Lyamichev et al., 1993, Science 260: 778).

Alternatively, the 5′-3′ exonuclease activity of a DNA polymerase isassayed using uniformly-labeled double-stranded DNA that is also nicked.The release of radioactivity (TCA soluble cpms) by a DNA polymerase inthe absence and presence of dNTPs is measured. Non-proofreading DNApolymerases with 5′-3′ exonuclease activity are stimulated 10-fold ormore by concomitant polymerization that occurs in the presence of dNTPs(increase in cpms released in the presence of dNTPs). Proofreading DNApolymerases with 3′-5′ exo activity are inhibited completely byconcomitant polymerization that occurs in the presence of dNTPs(decrease in cpms released in the presence of dNTPs) (U.S. Pat. No.5,352,778).

Nucleases useful according to the invention include any enzyme thatpossesses 5′ endonucleolytic activity for example a DNA polymerase, e.g.DNA polymerase I from E. coli, and DNA polymerase from Thermus aquaticus(Taq), Thermus thermophilus (Tth), and Thermus flavus (Tfl). Nucleasesuseful according to the invention also include DNA polymerases with5′-3′ exonuclease activity, including but not limited to eubacterial DNApolymerase I, including enzymes derived from Thermus species (Taq, Tfl,Tth, Tca (caldophilus) Thr (brockianus)), enzymes derived from Bacillusspecies (Bst, Bca, Magenta (full length polymerases, NOT N-truncatedversions)), enzymes derived from Thermotoga species (Tma (maritima, Tne(neopolitana)) and E. coli DNA polymerase I. The term nuclease alsoembodies FEN nucleases. Additional nucleic acid polymerases usefulaccording to the invention are included below in the section entitled,“Nucleic Acid Polymerases”

As used herein, “cleaving” refers to enzymatically separating a cleavagestructure into distinct (i.e. not physically linked to other fragmentsor nucleic acids by phosphodiester bonds) fragments or nucleotides andfragments that are released from the cleavage structure. For example,cleaving a labeled cleavage structure refers to separating a labeledcleavage structure according to the invention and defined below, intodistinct fragments including fragments derived from an oligonucleotidethat specifically hybridizes with a target nucleic acid or wherein oneof the distinct fragments is a labeled nucleic acid fragment derivedfrom a target nucleic acid and/or derived from an oligonucleotide thatspecifically hybridizes with a target nucleic acid that can be detectedand/or measured by methods well known in the art and described hereinthat are suitable for detecting the labeled moiety that is present on alabeled fragment.

As used herein, “endonuclease” refers to an enzyme that cleaves bonds,preferably phosphodiester bonds, within a nucleic acid molecule. Anendonuclease according to the invention can be specific forsingle-stranded or double-stranded DNA or RNA.

As used herein, “exonuclease” refers to an enzyme that cleaves bonds,preferably phosphodiester bonds, between nucleotides one at a time fromthe end of a polynucleotide. An exonuclease according to the inventioncan be specific for the 5′ or 3′ end of a DNA or RNA molecule, and isreferred to herein as a 5′ exonuclease or a 3′ exonuclease.

As used herein a “flap” refers to a region of single stranded DNA thatextends from a double stranded nucleic acid molecule. A flap accordingto the invention is preferably between about 1-10,000 nucleotides, morepreferably between about 5-25 nucleotides and most preferably betweenabout 10-20 nucleotides.

In a preferred embodiment, the binding moiety is a tag.

In another preferred embodiment, the binding moiety is a nucleic acidsequence that binds to a capture element.

The invention also provides a method of detecting or measuring a targetnucleic acid comprising the steps of: forming a cleavage structure byincubating a sample containing a target nucleic acid with a probe havinga secondary structure that changes upon binding of the probe to thetarget nucleic acid and, the probe further comprising a binding moiety,cleaving the cleavage structure with a nuclease to release a nucleicacid fragment wherein the cleavage is performed at a cleavingtemperature, and the secondary structure of the probe when not bound tothe target nucleic acid is stable at or below the cleaving temperature;and detecting and/or measuring the amount of the fragment captured bybinding of the binding moiety to a capture element on a solid support asan indication of the presence of the target sequence in the sample.

As used herein, “detecting a target nucleic acid” or “measuring a targetnucleic acid” refers to determining the presence of a particular targetnucleic acid in a sample or determining the amount of a particulartarget nucleic acid in a sample as an indication of the presence of atarget nucleic acid in a sample. The amount of a target nucleic acidthat can be measured or detected is preferably about 1 molecule to 10²⁰molecules, more preferably about 100 molecules to 10¹⁷ molecules andmost preferably about 1000 molecules to 10¹⁴ molecules. According to oneembodiment of the invention, the detected nucleic acid is derived fromthe labeled 5′ end of a downstream probe of a cleavage structureaccording to the invention (for example C in FIG. 4), that is displacedfrom the target nucleic acid by the 3′ extension of an upstream probe ofa cleavage structure according to the invention (for example A of FIG.4). According to the present invention, a label is attached to the 5′end of the downstream probe (for example C in FIG. 4) comprising acleavage structure according to the invention. Alternatively, a label isattached to the 3′ end of the downstream probe and a quencher isattached to the 5′ flap of the downstream probe. According to theinvention, a label may be attached to the 3′ end of the downstream probe(for example C in FIG. 4) comprising a cleavage structure according tothe invention.

According to the invention, the downstream probe (for example C in FIG.4) may be labeled internally. In a preferred embodiment, a cleavagestructure according to the invention can be prepared by incubating atarget nucleic acid with a probe having a secondary structure thatchanges upon binding of the probe to the target nucleic acid, andfurther comprising a non-complementary, labeled, 5′ region that does notanneal to the target nucleic acid and forms a 5′ flap, and acomplementary 3′ region that anneals to the target nucleic acid.According to this embodiment of the invention, the detected nucleic acidis derived from the labeled 5′ flap region of the probe. Preferablythere is a direct correlation between the amount of the target nucleicacid and the signal generated by the cleaved, detected nucleic acid.

In another embodiment, the probe is labeled with a pair of interactivelabels (e.g., a FRET or non-FRET pair) positioned to permit theseparation of the labels during oligonucleotide probe unfolding (e.g.,for example due to a change in the secondary structure of the probe) orhydrolysis. As used herein, “detecting the amount of the fragmentcaptured by a capture element on a solid support” or “measuring theamount of the fragment captured by a capture element on a solid support”or “detecting the amount of the fragment captured by a capture elementon a solid support” or “measuring the amount of the fragment captured bya capture element on a solid support” refers to determining the presenceof a labeled or unlabeled fragment in a sample or determining the amountof a labeled or unlabeled fragment in a sample. Methods well known inthe art and described herein can be used to detect or measure release oflabeled or unlabeled fragments bound to a capture element on a solidsupport, or following the release of the labeled or unlabeled fragmentfrom a capture element on a solid support. The detection methodsdescribed herein are operative for detecting a fragment wherein anyamount of a fragment is detected whether that be a small or largeproportion of the fragments generated in the reaction. A method ofdetecting or measuring release of labeled fragments will be appropriatefor measuring or detecting the labeled moiety that is present on thelabeled fragments bound to a capture element on a solid support. Methodsof detecting or measuring release of unlabeled fragments include, forexample, gel electrophoresis or by hybridization, according to methodswell known in the art. The detection methods described herein areoperative when as little as 1 or 2 molecules (and up to 1 or 2 million,for example 10, 100, 1000, 10,000, 1 million) of released fragment aredetected.

As used herein, “labeled fragments” refer to cleaved mononucleotides orsmall oligonucleotides or oligonucleotides derived from the labeledcleavage structure according to the invention wherein the cleavedoligonucleotides are preferably between about 1-1000 nucleotides, morepreferably between about 5-50 nucleotides and most preferably betweenabout 16-18 nucleotides, which are cleaved from a cleavage structure bya nuclease and can be detected by methods well known in the art anddescribed herein.

In one embodiment, a probe is a bi-molecular or multimolecular probewherein a first molecule comprising the probe is labeled with afluorophore and a second molecule comprising the probe is labeled with aquencher. As used herein, a “subprobe” and “subquencher” refer to afirst molecule of a bi- or multi-molecular probe according to theinvention, that is labeled with a fluorophore and a second molecule of abi- or multi-molecular probe according to the invention, that is labeledwith a quencher, respectively. According to this embodiment, followingbinding of the bi- or multi-molecular probe to the target nucleic acid,and cleavage by a nuclease, the subprobe and subquencher dissociate fromeach other (that is, the distance between the subprobe and thesubquencher increases) and a signal is generated as a result of thisdissociation and subsequent separation of the subprobe and subquencher.

In a preferred embodiment, the binding moiety is a tag.

In another preferred embodiment, the binding moiety is a nucleic acidsequence that binds to a capture element.

In a preferred embodiment, the method further comprises a nucleic acidpolymerase.

In another preferred embodiment, the cleavage structure furthercomprises a 5′ flap.

In another preferred embodiment, the cleavage structure furthercomprises an oligonucleotide primer.

In another preferred embodiment, the secondary structure is selectedfrom the group consisting a stem-loop structure, a hairpin structure, aninternal loop, a bulge loop, a branched structure, a pseudoknotstructure or a cloverleaf structure.

In another preferred embodiment, the nuclease is a FEN nuclease.

In another preferred embodiment the FEN nuclease is selected from thegroup consisting of FEN nuclease enzyme derived from Archaeglobusfulgidus, Methanococcus jannaschii, Pyrococcus furiosus, human, mouse orXenopus laevis. A FEN nuclease according to the invention also includesSaccharomyces cerevisiae RAD27, and Schizosaccharomyces pombe RAD2, PolI DNA polymerase associated 5′ to 3′ exonuclease domain, (e.g. E. coli,Thermus aquaticus (Taq), Thermus flavus (Tfl), Bacillus caldotenax(Bca), Streptococcus pneumoniae) and phage functional homologs of FENincluding but not limited to T4, T5 5′ to 3′ exonuclease, T7 gene 6exonuclease and T3 gene 6 exonuclease.

Preferably, only the 5′ to 3′ exonuclease domains of Taq, Tfl and BcaFEN nuclease are used.

In another preferred embodiment, the probe further comprises a reporter.

In another preferred embodiment, the reporter comprises a tag.

In another preferred embodiment, the fragment is captured by binding ofthe tag to a capture element.

In another preferred embodiment, the cleavage structure is formedcomprising at least one labeled moiety capable of providing a signal.

In another preferred embodiment, the cleavage structure is formedcomprising a pair of interactive signal generating labeled moietieseffectively positioned on the probe to quench the generation of adetectable signal when the probe is not bound to the target nucleicacid.

In another preferred embodiment, the labeled moieties are separated by asite susceptible to nuclease cleavage, thereby allowing the nucleaseactivity of the nuclease to separate the first interactive signalgenerating labeled moiety from the second interactive signal generatinglabeled moiety by cleaving at the site susceptible to nuclease cleavage,thereby generating a detectable signal.

The presence of a pair of interactive signal generating labeledmoieties, as described above, allows for discrimination betweenannealed, uncleaved probe that may bind to a capture element, andreleased labeled fragment that is bound to a capture element.

In another preferred embodiment, the pair of interactive signalgenerating moieties comprises a quencher moiety and a fluorescentmoiety.

The invention also provides for a polymerase chain reaction process fordetecting a target nucleic acid in a sample. This process comprises,providing a cleavage structure comprising a probe having a secondarystructure that changes upon binding of the probe to the target nucleicacid and, the probe further comprising a binding moiety, a set ofoligonucleotide primers wherein a first primer contains a sequencecomplementary to a region in one strand of the target nucleic acid andprimes the synthesis of a complementary DNA strand, and a second primercontains a sequence complementary to a region in a second strand of thetarget nucleic acid and primes the synthesis of a complementary DNAstrand. This process also comprises amplifying the target nucleic acidemploying a nucleic acid polymerase as a template-dependent polymerizingagent under conditions which are permissive for PCR cycling steps of (i)annealing of primers required for amplification to a template nucleicacid sequence contained within the target nucleic acid, (ii) extendingthe primers providing that the nucleic acid polymerase synthesizes aprimer extension product, and (iii) cleaving the cleavage structureemploying a nuclease as a cleavage agent for release of labeledfragments from the cleavage structure thereby creating detectablelabeled fragments. According to this process, the cleaving is performedat a cleaving temperature and the secondary structure of the secondprimer when not bound to the target nucleic acid is stable at or belowthe cleaving temperature. The amount of released, labeled fragmentcaptured by binding of the binding moiety to a capture element on asolid support is detected and/or measured as an indicator of thepresence of the target sequence in the sample.

As used herein, an “oligonucleotide primer” refers to a single strandedDNA or RNA molecule that is hybridizable to a nucleic acid template andprimes enzymatic synthesis of a second nucleic acid strand.Oligonucleotide primers useful according to the invention are betweenabout 6 to 100 nucleotides in length, preferably about 17-50 nucleotidesin length and more preferably about 17-45 nucleotides in length.Oligonucleotide probes useful for the formation of a cleavage structureaccording to the invention are between about 17-40 nucleotides inlength, preferably about 17-30 nucleotides in length and more preferablyabout 17-25 nucleotides in length.

As used herein, “template dependent polymerizing agent” refers to anenzyme capable of extending an oligonucleotide primer in the presence ofadequate amounts of the four deoxyribonucleoside triphosphates (dATP,dGTP, dCTP and dTTP) or analogs as described herein, in a reactionmedium comprising appropriate salts, metal cations, appropriatestabilizers and a pH buffering system. Template dependent polymerizingagents are enzymes known to catalyze primer- and template-dependent DNAsynthesis, and possess 5′ to 3′ nuclease activity. Preferably, atemplate dependent polymerizing agent according to the invention lacks5′ to 3′ nuclease activity.

As used herein, “amplifying” refers to producing additional copies of anucleic acid sequence, including the method of the polymerase chainreaction.

In a preferred embodiment, the nuclease is a FEN nuclease.

In a preferred embodiment, the binding moiety is a tag.

In another preferred embodiment, the binding moiety is a nucleic acidsequence that binds to a capture element.

In another preferred embodiment, the oligonucleotide primers of step bare oriented such that the forward primer is located upstream of thecleavage structure and the reverse primer is located downstream of thecleavage structure.

In another preferred embodiment, the nucleic acid polymerase has stranddisplacement activity.

Nucleic acid polymerases exhibiting strand displacement activity anduseful according to the invention include but are not limited toarchaeal DNA polymerases with “temperature activated” stranddisplacement activity (exo plus and exo minus versions of Vent, DeepVent, Pfu, JDF-3, KOD (LTI's tradename Pfx), Pwo, 9 degrees North,Thermococcus aggregans, Thermococcus gorgonarius), and eubacterial DNApolymerases with strand displacement activity (exo minus Bst, exo minusBca, Genta, Klenow fragment, exo minus Klenow fragment exo minus T7 DNApolymerase (Sequenase).

In another preferred embodiment, the nucleic acid polymerase isthermostable In another preferred embodiment, the nuclease isthermostable.

As used herein, “thermostable” refers to an enzyme which is stable andactive at temperatures as great as preferably between about, 90-100° C.and more preferably between about 70-980 C to heat as compared, forexample, to a non-thermostable form of an enzyme with a similaractivity. For example, a thermostable nucleic acid polymerase or FENnuclease derived from thermophilic organisms such as P. furiosus, M.jannaschii, A. fulgidus or P. horikoshii are more stable and active atelevated temperatures as compared to a nucleic acid polymerase from E.coli or a mammalian FEN enzyme. A representative thermostable nucleicacid polymerase isolated from Thermus aquaticus (Taq) is described inU.S. Pat. No. 4,889,818 and a method for using it in conventional PCR isdescribed in Saiki et al., 1988, Science 239:487. Another representativethermostable nucleic acid polymerase isolated from P. furiosus (Pfu) isdescribed in Lundberg et al., 1991, Gene, 108:1-6. Additionalrepresentative temperature stable polymerases include, e.g., polymerasesextracted from the thermophilic bacteria Thermus flavus, Thermus ruber,Thermus thermophilus, Bacillus stearothermophilus (which has a somewhatlower temperature optimum than the others listed), Thermus lacteus,Thermus rubens, Thermotoga maritima, or from thermophilic archaeaThermococcus litoralis, and Methanothermus fervidus.

Temperature stable polymerases and FEN nucleases are preferred in athermocycling process wherein double stranded nucleic acids aredenatured by exposure to a high temperature (about 950 C) during the PCRcycle.

In another preferred embodiment, the nuclease is a flap-specificnuclease.

In another preferred embodiment, the probe further comprises a reporter.

In another preferred embodiment, the reporter comprises a tag.

In another preferred embodiment, the fragment is captured by binding ofsaid tag to a capture element.

In another preferred embodiment, the cleavage structure is formedcomprising at least one labeled moiety capable of providing a signal.

In another preferred embodiment, the cleavage structure is formedcomprising a pair of interactive signal generating labeled moietieseffectively positioned on the probe to quench the generation of adetectable signal when the probe is not bound to the target nucleicacid.

In another preferred embodiment, the labeled moieties are separated by asite susceptible to nuclease cleavage, thereby allowing the nucleaseactivity of the nuclease to separate the first interactive signalgenerating labeled moiety from the second interactive signal generatinglabeled moiety by cleaving at the site susceptible to nuclease cleavage,thereby generating a detectable signal.

In another preferred embodiment, the pair of interactive signalgenerating moieties comprises a quencher moiety and a fluorescentmoiety.

In another preferred embodiment, the nucleic acid polymerase is selectedfrom the group consisting of Taq polymerase and Pfu polymerase.

The invention provides for a polymerase chain reaction process whereinamplification and detection of a target nucleic acid occur concurrently(i.e., real time detection). The invention also provides for apolymerase chain reaction process wherein amplification of a targetnucleic acid occurs prior to detection of the target nucleic acid (i.e.,end point detection).

The invention also provides for a polymerase chain reaction process forsimultaneously forming a cleavage structure, amplifying a target nucleicacid in a sample and cleaving the cleavage structure. This processcomprises the step of: (a) providing an upstream oligonucleotide primercomplementary to a first region in one strand of the target nucleicacid, a downstream labeled probe complementary to a second region in thesame strand of the target nucleic acid, wherein the downstream labeledprobe is capable of forming a secondary structure that changes uponbinding of the probe to the target nucleic acid and, the probe furthercomprises a binding moiety, and a downstream oligonucleotide primercomplementary to a region in a second strand of the target nucleic acid.According to this step of the process, the upstream primer primes thesynthesis of a complementary DNA strand, and the downstream primerprimes the synthesis of a complementary DNA strand. This process alsocomprises the step of (b) detecting a nucleic acid which is produced andcaptured by binding of the binding moiety to a capture element on asolid support. The nucleic acid that is detected is produced in areaction comprising amplification and cleavage of the target nucleicacid wherein a nucleic acid polymerase is a template-dependentpolymerizing agent under conditions which are permissive for PCR cyclingsteps of (i) annealing of primers to a target nucleic acid, (ii)extending the primers of step (a), providing that the nucleic acidpolymerase synthesizes primer extension products, and the primerextension product of the upstream primer of step (a) partially displacesthe downstream probe of step (a) to form a cleavage structure. Theconditions are also permissive for (iii) cleaving the cleavage structureemploying a nuclease as a cleavage agent for release of detectablelabeled fragments from the cleavage structure. The cleaving is performedat a cleaving temperature and the secondary structure of the probe whennot bound to the target nucleic acid is stable at or below the cleavingtemperature.

In a preferred embodiment, the cleavage structure further comprises a 5′flap.

The invention also provides a method of forming a cleavage structurecomprising the steps of: (a) providing a target nucleic acid, (b)providing an upstream primer complementary to the target nucleic acid,(c) providing a downstream probe having a secondary structure thatchanges upon binding of the probe to a target nucleic acid and, theprobe further comprises a binding moiety; and (d) annealing the targetnucleic acid, the upstream primer and the downstream probe. The cleavagestructure can be cleaved with a nuclease at a cleaving temperature. Thesecondary structure of the probe when not bound to the target nucleicacid is stable at or below the cleaving temperature.

In a preferred embodiment, the cleavage structure comprises a 5′ flap.

The invention also provides for a composition comprising a targetnucleic acid, a probe having a secondary structure that changes uponbinding of the probe to a target nucleic acid and, the probe furthercomprises a binding moiety, and a nuclease. The probe and the targetnucleic acid of this composition can bind to form a cleavage structurethat can be cleaved by the nuclease at a cleaving temperature. Thesecondary structure of the probe when not bound to the target nucleicacid is stable at or below the cleaving temperature.

In a preferred embodiment, the composition further comprises anoligonucleotide primer.

In another preferred embodiment, the probe and the oligonucleotidehybridize to non-overlapping regions of the target nucleic acid.

The invention also provides for a kit for generating a signal indicativeof the presence of a target nucleic acid in a sample, comprising a probehaving a secondary structure that changes upon binding of the probe to atarget nucleic acid and, the probe further comprising a binding moiety,and a nuclease. The probe of this kit can bind to a target nucleic acidto form a cleavage structure that can be cleaved by the nuclease at acleaving temperature. The secondary structure of the probe when notbound to the target nucleic acid is stable at or below the cleavingtemperature.

In a preferred embodiment, the kit further comprises an oligonucleotideprimer.

In another preferred embodiment, the nuclease is a FEN nuclease.

In another preferred embodiment, the probe comprises at least onelabeled moiety.

In another preferred embodiment, the probe comprises a pair ofinteractive signal generating labeled moieties effectively positioned toquench the generation of a detectable signal when the probe is not boundto the target nucleic acid.

In another preferred embodiment, the labeled moieties are separated by asite susceptible to nuclease cleavage, thereby allowing the nucleaseactivity of the nuclease to separate the first interactive signalgenerating labeled moiety from the second interactive signal generatinglabeled moiety by cleaving at the site susceptible to nuclease cleavage,thereby generating a detectable signal.

In another preferred embodiment, the pair of interactive signalgenerating moieties comprises a quencher moiety and a fluorescentmoiety.

Further features and advantages of the invention are as follows. Theclaimed invention provides a method of generating a signal to detectand/or measure a target nucleic acid wherein the generation of a signalis an indication of the presence of a target nucleic acid in a sample.The method of the claimed invention does not require multiple steps. Theclaimed invention also provides a PCR based method for detecting and/ormeasuring a target nucleic acid comprising generating a signal as anindication of the presence of a target nucleic acid. The claimedinvention allows for simultaneous amplification and detection and/ormeasurement of a target nucleic acid. The claimed invention alsoprovides a PCR based method for detecting and/or measuring a targetnucleic acid comprising generating a signal in the absence of a nucleicacid polymerase that demonstrates 5′ to 3′ exonuclease activity.

Further features and advantages of the invention will become more fullyapparent in the following description of the embodiments and drawingsthereof, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 demonstrates FEN nuclease cleavage structures.

FIG. 2 demonstrates three templates (labeled 1, 2, and 3) that may beused to detect FEN nuclease activity.

FIG. 3 demonstrates secondary structures.

FIG. 4 is a diagram illustrating a synthesis and cleavage reaction togenerate a signal according to the invention.

FIG. 5 is a Sypro Orange stained polyacrylamide gel demonstratingCBP-tagged PFU FEN-1 protein.

FIG. 6 is an autoradiograph of a FEN-1 nuclease assay.

FIG. 7 is a representation of an open circle probe for rolling circleamplification.

FIG. 8 is a representation of rolling circle amplification.

FIG. 9 is a representation of a safety pin probe.

The sequence tcgcagtgtc gacctgcgc is SEQ ID NO: 31

The sequence cagccgtcga tccgcaggtc gacactgccg tcgacggctg is SEQ ID NO:32

The sequence gcagctgccg ac is SEQ ID NO: 33

The sequence tccgcaggtc gacactgccg tcgacggctg is SEQ ID NO: 34

FIG. 10 is a representation of a scorpion probe.

FIG. 11 is a representation of a sunrise/amplifluor probe

FIG. 12 a is a graph demonstrating the difference in light absorbance ofdouble-stranded versus single-stranded DNA.

FIG. 12 b is a graph demonstrating DNA melting curves.

FIG. 12 c is a graph demonstrating the effects of temperature on therelative optical absorbance of DNA.

FIG. 12 d is a graph demonstrating the effects of temperature on therelative optical absorbance of DNA.

FIG. 12 e is a graph demonstrating the effects of temperature on thefluorescence of DNA labeled with a pair of interactive labels.

FIG. 12 f is a graph demonstrating the effects of temperature on thefluorescence of DNA labeled with a pair of interactive labels.

FIG. 12 g is a graph demonstrating the effects of a target nucleic acidon the fluorescence of DNA labeled with a pair of interactive labels.

DESCRIPTION

The invention provides for compositions and methods of generating asignal to detect the presence of a target nucleic acid in a samplewherein a nucleic acid is treated with the combination of a probe, anRNA polymerase and a nuclease. The invention also provides for a processfor detecting or measuring a nucleic acid that allows for concurrentamplification, cleavage and detection of a target nucleic acid in asample.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: A LaboratoryManual, Second Edition; Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Nucleic Acid Hybridization (B. D. Harnes & S. J. Higgins, eds.,1984); A Practical Guide to Molecular Cloning (B. Perbal, 1984); and aseries, Methods in Enzymology (Academic Press, Inc.); Short Protocols InMolecular Biology, (Ausubel et al., ed., 1995). All patents, patentapplications, and publications mentioned herein, both supra and infra,are hereby incorporated by reference.

I. Nucleases

Nucleases useful according to the invention include any enzyme thatpossesses 5′ endonucleolytic activity for example a DNA polymerase, e.g.DNA polymerase I from E. coli, and DNA polymerase from Thermus aquaticus(Taq), Thermus thermophilus (Tth), and Thermus flavus (Tfl). Nucleasesuseful according to the invention also include DNA polymerases with5′-3′ exonuclease activity, including but not limited to eubacterial DNApolymerase I, including enzymes derived from Thermus species (Taq, Tfl,Tth, Tca (caldophilus) Thr (brockianus)), enzymes derived from Bacillusspecies (Bst, Bca, Magenta (full length polymerases, NOT N-truncatedversions)), enzymes derived from Thermotoga species (Tma (maritima, Tne(neopolitana)) and E. coli DNA polymerase I. The term nuclease alsoembodies FEN nucleases. A nuclease useful according to the inventioncannot cleave either a probe or primer that is not hybridized to atarget nucleic acid or a target nucleic acid that is not hybridized to aprobe or a primer.

FEN-1 is an ˜40 kDa divalent metal ion-dependent exo- and endonucleasethat specifically recognizes the backbone of a 5′ single-stranded flapstrand and tracks down this arm to the cleavage site, which is locatedat the junction wherein the two strands of duplex DNA adjoin thesingle-stranded arm. Both the endo- and exonucleolytic activities showlittle sensitivity to the base at the most 5′ position at the flap ornick. Both FEN-1 endo- and exonucleolytic substrate binding and cuttingare stimulated by an upstream oligonucleotide (flap adjacent strand orprimer). This is also the case for E. coli pol I. The endonucleaseactivity of the enzyme is independent of the 5′ flap length, cleaving a5′ flap as small as one nucleotide. The endonuclease and exonucleaseactivities are insensitive to the chemical nature of the substrate,cleaving both DNA and RNA.

Both the endo- and exonucleolytic activities are inhibited byconcentrations of salts in the physiological range. The exonucleaseactivity is inhibited 50-fold at 50 mM NaCl as compared to 0 mM NaCl.The endonuclease activity is inhibited only sevenfold at 50 mM NaCl(Reviewed in Lieber 1997, supra).

Although a 5′-OH terminus is a good substrate for FEN-1 loading onto a5′ flap substrate, it serves as a very poor substrate when part of anick in an otherwise double stranded DNA structure. The electrostaticrepulsion by the terminal phosphate is likely to favor breathing of thesubstrate into a pseudo-flap configuration, providing the active form ofthe substrate for FEN-1. Such an explanation would indicate a singleactive site and a single mechanism of loading of FEN-1 onto the 5′ ssDNAterminus of the flap or pseudo-flap configuration of the nick.Consistent with this model are observations that optimal activity at anick requires very low Mg²⁺ and monovalent salt concentrations, whichdestabilize base-pairing and would favor breathing of a nick to a flap.Higher Mg²⁺ and monovalent salt concentrations would disfavor breathingand inhibit cutting of nicked or gapped structures that do requirebreathing to convert to a flap. Cleavage of stable flap structures isoptimal at moderate Mg²⁺ levels and does not decrease with increasingMg²⁺ concentration. This is because a flap substrate does not have tomelt out base pairs to achieve its structure; hence, it is entirelyinsensitive to Mg²⁺. Though the endonucleolytic activity decreases withmonovalent salt, the decline is not nearly as sharp as that seen for theexonucleolytic activity. Furthermore, it has previously been shown thatone-nucleotide flaps are efficient substrates. All of these observationsare consistent with the fact that when FEN-1 has been interpreted to befunctioning as an exonuclease, the size of the degradation products varyfrom one to several nucleotides in length. Breathing of nicks into flapsof varying length would be expected to vary with local sequence,depending on the G/C content. In summary, a nick breathing to form atransient flap means that the exonucleolytic activity of FEN-1 is thesame as the endonucleolytic activity (Reviewed in Lieber, 1997, supra).

The endonuclease and exonuclease activities of FEN-1 cleave both DNA andRNA without requiring accessory proteins. At the replication fork,however, FEN-1 does interact with other proteins, including a DNAhelicase and the proliferating cell nuclear antigen (PCNA), theprocessivity factor for DNA polymerases δ and ε. PCNA significantlystimulates FEN-1 endo- and exonucleolytic activity.

The FEN-1 enzymes are functionally related to several smallerbacteriophage 5′→3′ exonucleases such as T5 5′ exonuclease and T4 RNaseH as well as to the larger eukaryotic nucleotide excision repair enzymessuch as XPG, which also acts in the transcription-coupled repair ofoxidative base damage. In eubacteria such as Escherichia coli andThermus aquaticus, Okazaki processing is provided by the PolI 5′→3′exonuclease domain. These bacterial and phage enzymes share two areas oflimited sequence homology with FEN-1, which are termed the N(N-terminal)and I (intermediate) regions, with the residue similarities concentratedaround seven conserved acidic residues. Based on crystal structures ofT4 RNase H and T5 exonuclease as well as mutagenesis data, it has beenproposed that these residues bind to two Mg²⁺ ions that are required foraffecting DNA hydrolysis; however, the role each metal plays in thecatalytic cycle, which is subtly different for each enzyme, is not wellunderstood (Reviewed in Hosfield et al., 1998b, supra).

Fen-1 genes encoding FEN-1 enzymes useful in the invention includemurine fen-1, human fen-1, rat fen-1, Xenopus laevis fen-1, and fen-1genes derived from four archaebacteria Archaeglobus fulgidus,Methanococcus jannaschii, Pyrococcus furiosus and Pyrococcus horikoshii.cDNA clones encoding FEN-1 enzymes have been isolated from human(GenBank Accession Nos.: NM_(—)004111 and L37374), mouse (GenBankAccession No.: L26320), rat (GenBank Accession No.: AA819793), Xenopuslaevis (GenBank Accession Nos.: U68141 and U64563), and P. furiosus(GenBank Accession No.: AF013497). The complete nucleotide sequence forP. horikoshii flap endonuclease has also been determined (GenBankAccession No.: AB005215). The FEN-1 family also includes theSaccharomyces cerevisiae RAD27 gene (GenBank Accession No.: Z28113Y13137) and the Saccharomyces pombe RAD2 gene (GenBank Accession No.:X77041). The archaeal genome of Methanobacterium thermautotrophiculumhas also been sequenced. Although the sequence similarity between FEN-1and prokaryotic and viral 5′→3′ exonucleases is low, FEN-1s within theeukaryotic kingdom are highly conserved at the amino acid level, withthe human and S. cerevisiae proteins being 60% identical and 78%similar. The three archaebacterial FEN-1 proteins are also, highlyhomologous to the eukaryotic FEN-1 enzymes (Reviewed in Matsui et al.,1999., J. Biol. Chem., 274:18297, Hosfield et al., 1998b, J. Biol.Chem., 273:27154 and Lieber, 1997, BioEssays, 19:233).

The sequence similarities in the two conserved nuclease domains(N-terminal or N and intermediate or I domains) between human and otherFEN-1 family members are 92% (murine), 79% (S. cerevisiae), 77% (S.pombe), 72% (A. fulgidus), 76% (M. jannaschii), and 74% (P. furiosus).

FEN-1 specifically recognizes the backbone of a 5′ single-stranded flapstrand and migrates down this flap arm to the cleavage site located atthe junction between the two strands of duplex DNA and thesingle-stranded arm. If the strand upstream of the flap (sometimescalled the flap adjacent strand or primer strand) is removed, theresulting structure is termed a pseudo-Y (see FIG. 1). This structure iscleaved by FEN-1, but at 20- to 100-fold lower efficiency. FEN-1 doesnot cleave 3′ single-stranded flaps. However, FEN-1 acting as anexonuclease will hydrolyze dsDNA substrates containing a gap or nick(Reviewed in Hosfield et al., 1998a, supra, Hosfield et al., 1999b,supra and Lieber 1997, supra). Exonucleolytically, FEN-1 acts at a nickand, with lower efficiency, at a gap or a recessed 5′ end on dsDNA. Atgapped structures, the efficiency of FEN-1 binding and cutting decreaseswith increasing gap size up to approximately five nucleotides and thenstabilizes at a level of cleavage that is equivalent to activity on arecessed 5′ end within dsDNA. Blunt dsDNA, recessed 3′ ends and ssDNAare not cleaved (Reviewed in Lieber 1997, supra). The cleavage activityof FEN enzymes are described in Yoon et al., 1999, Biochemistry, 38:4809; Rao, 1998, J. Bacteriol., 180:5406 and Hosfield et al., 1998,Cell, 95:135-146, incorporated herein by reference.

FEN nucleases that are useful according to the invention have beenisolated from a variety of organisms including human (GenBank AccessionNos.: NM_(—)004111 and L37374), mouse (GenBank Accession No.: L26320),rat (GenBank Accession No.: AA819793), yeast (GenBank Accession No.:Z28113 Y13137 and GenBank Accession No.: X77041) and xenopus laevis(GenBank Accession Nos.: U68141 and U64563). Such enzymes can be clonedand overexpressed using conventional techniques well known in the art.

A FEN nuclease according to the invention is preferably thermostable.Thermostable FEN nucleases have been isolated and characterized from avariety of thermostable organisms including four archeaebacteria. ThecDNA sequence (GenBank Accession No.: AF013497) and the amino acidsequence (Hosfield et al., 1998a, supra and Hosfield et al., 1998b) forP. furiosus flap endonuclease have been determined. The completenucleotide sequence (GenBank Accession No.: AB005215) and the amino acidsequence (Matsui et al., supra) for P. horikoshii flap endonuclease havealso been determined. The amino acid sequence for M. jannaschii(Hosfield et al., 1998b and Matsui et al., 1999 supra) and A. fulgidus(Hosfield et al., 1998b) flap endonuclease have also been determined.

Thermostable FEN1 enzymes can be cloned and overexpressed usingtechniques well known in the art and described in Hosfield et al.,1998a, supra, Hosfield et al., 1998b; Kaiser et al., 1999, J. Biol.Chem., 274: 21387 and Matusi et al., supra and herein in Example 2entitled “Cloning Pfu FEN-1”.

The endonuclease activity of a FEN enzyme can be measured by a varietyof methods including the following.

A. Fen Endonuclease Activity Assay

1. Templates (for example as shown in FIG. 2) are used to evaluate theactivity of a FEN nuclease according to the invention.

Template 1 is a 5′ ³³P labeled oligonucleotide (Heltest4) with thefollowing sequence: 5′AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG 3′(SEQ ID NO: 1). The underlined section of Heltest4 represents the regioncomplementary to M13 mp18+. The cleavage product is an 18 nucleotidefragment with the sequence AAAATAAATAAAAAAAAT (SEQ ID NO: 2).

Heltest4 binds to M13 to produce a complementary double stranded domainas well as a non-complementary 5′ overhang. This duplex forms template 2(FIG. 2) which is also used for helicase assays. Template 3 (FIG. 2) hasan additional primer (FENAS) bound to M13 and is directly adjacent toHeltest 4. The sequence of FENAS is: 5′CCATTCGCCATTCAGGCTGCGCA 3′ (SEQID NO: 3). In the presence of template 3, FEN binds the free 5′ terminusof Heltest4, migrates to the junction and cleaves Heltest4 to produce an18 nucleotide fragment. Templates 1 and 2 serve as controls, althoughtemplate 2 can also serve as a template.

Templates are prepared as described below:

Template 1 Template 2 Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl14 μl FENAS ** ** 14 μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl  4.6 μl 4.6 μl 

10× Pfu buffer is available from Stratagene (Catalog #200536). Accordingto the method of the invention, 10× Pfu buffer is diluted such that areaction is carried out in the presence of 1× buffer.

M13 is M13 mp18+ strand and is at a concentration of 200 ng/μL, ³³Plabeled Heltest4 is at an approximate concentration of 0.7 ng/μl, andFENAS is at a concentration of 4.3 ng/μl. Based on these concentrations,the Heltest4 and M13 are at approximately equal molar amounts (5×10⁻¹⁴)and FENAS is present in an approximately 10× molar excess (6×10⁻¹³).

The template mixture is heated at 950 C for five minutes, cooled to roomtemperature for 45 minutes and stored at 40 C overnight.

2 μl of FEN-1 or, as a control, H₂O are mixed with the three templatesas follows:

  3 μl template  0.7 μl 10x cloned Pfu buffer 0.56 μl 100 mM MgCl₂ 2.00μl enzyme or H₂O 0.74 μl H₂O 7.00 μl total volume

The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide, 7M urea CastAway (Stratagene) gel.

Alternatively, FEN activity can be analyzed in the following bufferwherein a one hour incubation time is utilized.

10× FEN Buffer 500 mM Tris-HCl pH 8.0 100 mM MgCl₂

The reaction mixture below is mixed with 2 μl of FEN or, as a control, 2μl of H₂O.

  3 μl template  0.7 μl 10x FEN buffer 2.00 μl enzyme or H₂O  1.3 μl H₂O7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution, samples are heated at 99° C. for five minutes. Samples areloaded on an eleven-inch long, hand-poured, 20% acrylamide/bisacrylamide, 7M urea gel. The gel is run at 20 watts until thebromophenol blue has migrated approximately ⅔ the total distance. Thegel is removed from the glass plates and soaked for 10 minutes in fix(15% methanol, 5% acetic acid) and then for 10 minutes in water. The gelis placed on Whatmann 3 mm paper, covered with plastic wrap and driedfor 2 hours in a heated vacuum gel dryer. The gel is exposed overnightto X-ray film.

2. FEN endonuclease activity can also be measured according to themethod of Kaiser et al., supra). Briefly, reactions are carried out in a101 volume containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05% NonidetP-40, 10 g/ml tRNA, and 200 mM KCl for TaqPol and TthPol or 50 mM KClfor all other enzymes. Reaction conditions can be varied depending onthe cleavage structure being analyzed. Substrates (21M) and varyingamounts of enzyme are mixed with the indicated (above) reaction bufferand overlaid with Chill-out (MJ Research) liquid wax. Substrates areheat denatured at 900 C for 20 s and cooled to 500 C, then reactions arestarted by addition of MgCl₂ or MnCl₂ and incubated at 500 C for thespecified length of time. Reactions are stopped by the addition of 10 μlof 95% formamide containing 10 mM EDTA and 0.02% methyl violet (Sigma).

Samples are heated to 90° C. for 1 min immediately beforeelectrophoresis on a 20% denaturing acrylamide gel (19:1 cross-linked),with 7M urea, and in a buffer of 45 mM Tris borate, pH 8.3, 1.4 mM EDTA.Unless otherwise indicated, 1 μl of each stopped reaction is loaded perlane. Gels are scanned on an FMBIO-100 fluorescent gel scanner (Hitachi)using a 505-nm filter. The fraction of cleaved product is determinedfrom intensities of bands corresponding to uncut and cut substrate withFMBIO Analysis software (version 6.0, Hitachi). The fraction of cutproduct should not exceed 20% to ensure that measurements approximateinitial cleavage rates. The cleavage rate is defined as theconcentration of cut product divided by the enzyme concentration and thetime of the reaction (in minutes). For each enzyme three data points areused to determine the rate and experimental error.

3. FEN endonuclease activity can also be measured according to themethod of Hosfield et al., 1998a, supra. Briefly, in a final volume of13 μl, varying amounts of FEN and 1.54 μmol of labeled cleavagesubstrate are incubated at different temperatures for 30 min before thereaction is quenched with an equal volume of stop solution (10 mM EDTA,95% deionized formamide, and 0.008% bromophenol blue and xylene cyanol).Samples are electrophoresed through denaturing 15% polyacrylamide gels,and the relative amounts of starting material and product arequantitated using the IPLabGel system (Stratagene) running MacBAS imageanalysis software. Most reactions are performed in standard assay buffer(10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, and 50 μg/ml bovine serumalbumin); however, in a series of experiments the effect of differentdivalent metals and pH levels are studied by varying the standardbuffer. For divalent metals, MgCl₂ is omitted, and different metal ionsare used at a final concentration of 10 mM. To study the influence ofpH, buffers containing different amounts of Tris-HCl, glycine, andsodium acetate are used at a final concentration of 10 mM to obtain awide range of pH levels at 250 C.

4. FEN endonuclease activity can also be measured according to themethod of Matusi et al., 1999, supra. Briefly, the enzyme reactions areperformed in a 15-μl reaction mixture containing 50 mMTris-HCl (pH 7.4),1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol, 100 μg/ml bovine serum albumin,and 0.6 pmol of a labeled cleavage structure. After incubation for 30min at 600 C, the reaction is terminated by adding 15 μl of 95%formamide containing 10 mM EDTA and 1 mg/ml bromphenol blue. The samplesare heated at 950 C for 10 min, loaded onto a 15% polyacrylamide gel (35cm×42.5 cm) containing 7M urea and 10×TBE (89 mM Tris-HCl, 89 mM boricacid, 2 mM EDTA (pH 8.0)), and then electrophoresed for 2 h at 2000 V.Reaction products are visualized and quantified using a Phosphorlmager(Bio-Rad). Size marker, oligonucleotides are 5′ end-labeled with[γ-³²P]ATP and T4 polynucleotide kinase.

To determine the optimum pH, the reaction is performed in an assaymixture (15 μl) containing 1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol, 100μg/ml bovine serum albumin, and 0.6 pmol of 5′ end-labeled cleavagestructure in 50 mM of one of the following buffers at 600 C for 30 min.Three different 50 mM buffers are used to obtain a wide pH range asfollows: sodium acetate buffer (pH 4.0-5.5), phosphate buffer (pH5.5-8.0), and borate buffer (pH 8.0-9.4).

B. Fen Exonuclease Activity Assay

The exonuclease activity of a FEN nuclease according to the inventioncan be measured by the method of measuring FEN-1 endonuclease activitydescribed in Matsui et al., 1999, supra and summarized above.

Alternatively, the exonuclease activity of a FEN enzyme can be analyzedby the method described in Hosfield et al., 1998b, supra. Briefly,exonuclease activities are assayed using a nicked substrate of FEN underconditions identical to those described for the endonuclease assays(described above).

The precise positions of DNA cleavage in both the exonuclease andendonuclease experiments can be obtained by partial digestion of a 5′³²P-labeled template strand using the 3′-5′ exonuclease activity ofKlenow fragment.

A cleavage structure according to one embodiment of the inventioncomprises a partially displaced 5′ end of an oligonucleotide probeannealed to a target nucleic acid. Another cleavage structure accordingto the invention comprises a target nucleic acid (for example B in FIG.4), an upstream oligonucleotide probe according to the invention, andcomprising a region or regions that are complementary to the targetsequence (for example A in FIG. 4), and a downstream oligonucleotidethat is complementary to the target sequence (for example C in FIG. 4).A cleavage structure according to the invention can be formed by overlapbetween the upstream oligonucleotide and the downstream probe, or byextension of the upstream oligonucleotide by the synthetic activity of anucleic acid polymerase, and subsequent partial displacement of the 5′end of the downstream oligonucleotide. A cleavage structure of this typeis formed according to the method described in the section entitled“Cleavage Structure”.

Alternatively, a cleavage structure according to the invention is formedby annealing a target nucleic acid to an oligonucleotide probe accordingto the invention wherein the oligonucleotide probe comprises a region orregions that are complementary to the target nucleic acid, and anon-complementary region that does not anneal to the target nucleic acidand forms a 5′ flap. According to this embodiment, a cleavage structurecomprises a 5′ flap formed by a non-complementary region of theoligonucleotide.

A cleavage structure according to the invention also comprises anoverlapping flap wherein the 3′ end of an upstream oligonucleotidecapable of annealing to a target nucleic acid (for example A in FIG. 4)is complementary to 1 (or more) base pair of the downstreamoligonucleotide probe according to the invention (for example C in FIG.4) that is annealed to a target nucleic acid and wherein the 1 (or more)base pair overlap is directly downstream of the point of extension ofthe single stranded flap and is formed according to method described inthe section entitled “Cleavage Structure”. In one embodiment, theupstream oligonucleotide and the downstream probe hybridize tonon-overlapping regions of the target nucleic acid. In anotherembodiment, the upstream oligonucleotide and the downstream probehybridize to adjacent regions of the target nucleic acid.

II. Nucleic Acid Polymerases

The invention provides for nucleic acid polymerases. Preferably, thenucleic acid polymerase according to the invention is thermostable.

Known DNA polymerases useful according to the invention include, forexample, E. coli DNA polymerase I, Thermus thermophilus (Tth) DNApolymerase, Bacillus stearothermophilus DNA polymerase, Thermococcuslitoralis DNA polymerase, Thermus aquaticus (Taq) DNA polymerase andPyrococcus furiosus (Pfu) DNA polymerase.

Known RNA polymerases useful according to the invention include, forexample, include bacteriophage T7, T3 and SP6 RNA polymerases, E. coliRNA polymerase holoenzyme, E. coli RNA polymerase core enzyme, and humanRNA polymerase I, II, III, human mitochondrial RNA polymerase and NS5BRNA polymerase from (HCV).

Nucleic acid polymerases substantially lacking 5′ to 3′ exonucleaseactivity useful according to the invention include but are not limitedto Klenow and Klenow exo-, and T7 DNA polymerase (Sequenase).

Thermostable nucleic acid polymerases substantially lacking 5′ to 3′exonuclease activity useful according to the invention include but arenot limited to Pfu, exo-Pfu (a mutant form of Pfu that lacks 3′ to 5′exonuclease activity), the Stoffel fragment of Taq, N-truncated Bst,N-truncated Bca, Genta, JdF3 exo-, Vent, Vent exo- (a mutant form ofVent that lacks 3′ to 5′ exonuclease activity), Deep Vent, Deep Ventexo- (a mutant form of Deep Vent that lacks 3′ to 5′ exonucleaseactivity), U1Tma, ThermoSequenase and Thermus Thermostable RNAPolymerase.

Nucleic acid polymerases useful according to the invention include bothnative polymerases as well as polymerase mutants, which lack 5′ to 3′exonuclease activity. Nucleic acid polymerases useful according to theinvention can possess different degrees of thermostability. Preferably,a nucleic acid polymerase according to the invention exhibits stranddisplacement activity at the temperature at which it can extend anucleic acid primer. In a preferred embodiment of the invention, anucleic acid polymerase lacks both 5′ to 3′ and 3′ to 5′ exonucleaseactivity.

Additional nucleic acid polymerases substantially lacking 5′ to 3′exonuclease activity with different degrees of thermostability usefulaccording to the invention are listed below.

A. Bacteriophage DNA polymerases (Useful for 370 C Assays):

Bacteriophage DNA polymerases are devoid of 5′ to 3′ exonucleaseactivity, as this activity is encoded by a separate polypeptide.Examples of suitable DNA polymerases are T4, T7, and φ29 DNA polymerase.The enzymes available commercially are: T4 (available from many sourcese.g., Epicentre) and T7 (available from many sources, e.g. Epicentre forunmodified and USB for 3′ to 5′ exo⁻ T7 “Sequenase” DNA polymerase).

B. Archaeal DNA Polymerases:

There are 2 different classes of DNA polymerases which have beenidentified in archaea: 1. Family B/pol α type (homologs of Pfu fromPyrococcus furiosus) and 2. pol II type (homologs of P. furiosus DP1/DP22-subunit polymerase). DNA polymerases from both classes have been shownto naturally lack an associated 5′ to 3′ exonuclease activity and topossess 3′ to 5′ exonuclease (proofreading) activity. Suitable DNApolymerases (pol α or pol II) can be derived from archaea with optimalgrowth temperatures that are similar to the desired assay temperatures.Examples of suitable archaea include, but are not limited to:

1. Thermolabile (useful for 370 C assays)—e.g., Methanococcus voltae

2. Thermostable (useful for non-PCR assays)—e.g., Sulfolobussolfataricus, Sulfolobus acidocaldarium, Methanococcus jannaschi,Thermoplasma acidophilum. It is estimated that suitable archaea exhibitmaximal growth temperatures of ≦80-85° C. or optimal growth temperaturesof ≦70-80° C.

3. Thermostable (useful for PCR assays)—e.g., Pyrococcus species(furiosus, species GB-D, species strain KODI, woesii, abysii,horikdshii), Thermococcus species (litoralis, species 9° North-7,species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobusfulgidus. It is estimated that suitable archaea would exhibit maximalgrowth temperatures of ≧80-85° C. or optimal growth temperatures of≧70-80° C. Appropriate PCR enzymes from the archaeal pol α DNApolymerase group are commercially available, including KOD (Toyobo), Pfx(Life Technologies, Inc.), Vent (New England BioLabs), Deep Vent (NewEngland BioLabs), and Pwo (Boehringer-Mannheim).

Additional archaea related to those listed above are described in thefollowing references: Archaea: A Laboratory Manual (Robb, F. T. andPlace, A. R., eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1995 and Thermophilic Bacteria (Kristjansson, J. K., ed.)CRC Press, Inc., Boca Raton, Fla., 1992.

C. Eubacterial DNA polymerases:

There are 3 classes of eubacterial DNA polymerases, pol I, II, and III.Enzymes in the Pol I DNA polymerase family possess 5′ to 3′ exonucleaseactivity, and certain members also exhibit 3′ to 5′ exonucleaseactivity. Pol II DNA polymerases naturally lack 5′ to 3′ exonucleaseactivity, but do exhibit 3′ to 5′ exonuclease activity. Pol III DNApolymerases represent the major replicative DNA polymerase of the celland are composed of multiple subunits. The pol III catalytic subunitlacks 5′ to 3′ exonuclease activity, but in some cases 3′ to 5′exonuclease activity is located in the same polypeptide.

There are no commercial sources of eubacterial pol II and pol III DNApolymerases. There are a variety of commercially available Pol I DNApolymerases, some of which have been modified to reduce or abolish 5′ to3′ exonuclease activity. Methods used to eliminate 5′ to 3′ exonucleaseactivity of pol I DNA polymerases include:

-   -   mutagenesis (as described in Xu et al., 1997, J. Mol. Biol.,        268:284 and Kim et al., 1997, Mol. Cells, 7:468).    -   N-truncation by proteolytic digestion (as described in Klenow et        al., 197.1, Eur. J. Biochem., 22: 371), or    -   N-truncation by cloning and expressing as C-terminal fragments        (as described in Lawyer et al., 1993, PCR Methods Appl., 2:275).

As for archaeal sources, the assay-temperature requirements determinewhich eubacteria should be used as a source of a DNA polymerase usefulaccording to the invention (e.g., mesophiles, thermophiles,hyperthermophiles).

1. Mesophilic/thermolabile (Useful for 370 C Assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: pol II or the pol III catalytic subunit        from mesophilic eubacteria, such as Escherchia coli,        Streptococcus pneumoniae, Haemophilus influenza, Mycobacterium        species (tuberculosis, leprae)    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Pol I DNA polymerases for N-truncation or        mutagenesis can be isolated from the mesophilic eubacteria        listed above (Ci). A commercially-available eubacterial DNA        polymerase pol I fragment is the Klenow fragment (N-truncated E.        coli pol I; Stratagene).

2. Thermostable (Useful for non PCR assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: Pol II or the pol III catalytic subunit        from thermophilic eubacteria, such as Bacillus species (e.g.,        stearothermophilus, caldotenax, caldovelox)    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Suitable pol I DNA polymerases for        N-truncation or mutagenesis can be isolated from thermophilic        eubacteria such as the Bacillus species listed above.        Thermostable N-truncated fragments of B. stearothermophilus DNA        polymerase pol I are commercially available and sold under the        trade names Bst DNA polymerase I large fragment (Bio-Rad and        Isotherm DNA polymerase (Epicentre)). A C-terminal fragment of        Bacillus caldotenax pol I is available from Panvera (sold under        the tradename Ladderman).

3. Thermostable (Useful for PCR assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: Pol II or polIII catalytic subunit from        Thermus species (aquaticus, thermophilus, flavus, ruber,        caldophilus, filiformis, brokianus) or from Thermotoga maritima.        The catalytic pol III subunits from Thermus thermophilus and        Thermus aquaticus are described in Yi-Ping et al., 1999, J. Mol.        Evol., 48:756 and McHenry et al., 1997, J. Mol. Biol., 272:178.    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Suitable pol I DNA polymerases for        N-truncation or mutagenesis can be isolated from a variety of        thermophilic eubacteria, including Thermus species and        Thermotoga maritima (see above). Thermostable fragments of        Thermus aquaticus DNA polymerase pol I (Taq) are commercially        available and sold under the trade names KlenTaq1 (Ab Peptides),        Stoffel fragment (Perkin-Elmer), and ThermoSequenase (Amersham).        In addition to C-terminal fragments, 5′ to 3′ exonuclease. Taq        mutants are also commercially available, such as TaqFS        (Hoffman-LaRoche). In addition to 5′-3′ exonuclease⁻ versions of        Taq, an N-truncated version of Thermotoga maritima DNA        polymerase I is also commercially available (tradename UlTma,        Perkin-Elmer).

Additional eubacteria related to those listed above are described inThermophilic Bacteria (Kristjansson, J. K., ed.) CRC Press, Inc., BocaRaton, Fla., 1992.

D. Eukaryotic 5′ to 3′ Exonuclease⁻ DNA polymerases (Useful for 370 Cassays)

There are several DNA polymerases that have been identified ineukaryotes, including DNA pol α (replication/repair), δ (replication), ε(replication), β (repair) and γ (mitochondrial replication). EukaryoticDNA polymerases are devoid of 5′ to 3′ exonuclease activity, as thisactivity is encoded by a separate polypeptide (e.g., mammalian FEN-1 oryeast RAD2). Suitable thermolabile DNA polymerases may be isolated froma variety of eukaryotes (including but not limited to yeast, mammaliancells, insect cells, Drosophila) and eukaryotic viruses (e.g., EBV,adenovirus).

It is possible that DNA polymerase mutants lacking 3′-5′ exonuclease(proofreading) activity, in addition to lacking 5′ to 3′ exonucleaseactivity, could exhibit improved performance in FEN-based detectionstrategies. For example, reducing or abolishing inherent 3′ to 5′exonuclease activity may lower background signals by diminishingnon-specific exonucleolytic degradation of labeled probes. Three 3′ to5′ exonuclease motifs have been identified, and mutations in theseregions have been shown to abolish 3′ to 5′ exonuclease activity inKlenow, φ29, T4, T7, and Vent DNA polymerases, yeast Pol α, Pol β, andPol γ, and Bacillus subtilis Pol III (reviewed in Derbeyshire et al.,1995,_Methods. Enzymol. 262:363). Methods for preparing additional DNApolymerase mutants, with reduced or abolished 3′ to 5′ exonucleaseactivity, are well known in the art.

Commercially-available enzymes that lack both 5′ to 3′ and 3′ to 5′exonuclease activities include Sequenase (exo⁻ T7; USB), Pfu exo⁻(Stratagene), exo⁻ Vent (New England BioLabs), exo⁻ DeepVent (NewEngland BioLabs), exo. Klenow fragment (Stratagene), Bst (Bio-Rad),Isotherm (Epicentre), Ladderman (Panvera), KlenTaq1 (Ab Peptides),Stoffel fragment (Perkin-Elmer), ThermoSequenase (USB), and TaqFS(Hoffman-LaRoche).

If polymerases other than Pfu are used, buffers and extensiontemperatures are selected to allow for optimal activity by theparticular polymerase useful according to the invention. Buffers andextension temperatures useful for polymerases according to the inventionare know in the art and can also be determined from the Vendor'sspecifications.

E. RNA Polymerases

RNA polymerases useful in the invention include both DNA and RNAdependent RNA polymerases. RNA dependent RNA polymerases have beencharacterized that can synthesize new strands of RNA from a primedtemplate, from an unprimed template or from either a primed or unprimedtemplate. RNA polymerases have been identified that recognize andsynthesize RNA from single-stranded RNA templates in the absence ofprimer. Illustrative of this type of priming mechanism is an RNApolymerases of hepatitis C virus (HCV), termed NS5B. NS5B has beencharacterized as the RNA polymerases responsible for replicating the HCVRNA genome. NS5B has been demonstrated to catalyze the elongation of RNAsynthesis by either self-priming, extending an existing primer, orinitiating RNA synthesis de novo. Luo et al., J. Virol., 2000,74(2):851-853.

RNA polymerases have also been identified that recognize and synthesizeRNA from a primed single-stranded RNA template. Several referencesdetail RNA polymerases that can be used in this context, for example,Schiebel et al., J. Biol. Chem., 1993, 268(16):11858-11867, Tang et al.,Genes and Dev., 2003, 17(1):49-63, or U.S. patent application Ser. No.11/217,972, filed Aug. 31, 2005 (herein incorporated by reference in itsentirety) In these cases, the RNA polymerases can be used to selectivelyamplify a target population of RNA.

A “RNA-dependent RNA polymerase” is an enzyme that synthesizes multipleRNA copies from an RNA. Table 3 provides a non-exhaustive list of RNAdependent RNA polymerases that may be useful in performing theinvention. These polymerases and methods of using them to amplify atarget are described in U.S. patent application Ser. No. 11/217,972,filed Aug. 31, 2005 (herein incorporated by reference in its entirety).

TABLE 3 RNA POLYMERASES USEFUL IN INVENTION Source Primer MechanismCitations Bacteriophage Non-specific or US Patent Publication No. phi6-phl 14 Specific 20030124559, Jul. 3, 2003 Tomato U.S. Pat. No.6,218,142, US Patent Publication No. 20010023067, Sep. 20, 2001 Schiebelet. al. J. Biol. Chem (1993) 268: 11858 Tobacco — Ikegami et al. Proc.Natl. Acad. Sci USA (1978) 75: 2122 Cucumber — Khan et al., Proc. Natl.Acad. Sci. USA (1986) 83: 2383 Wheat Tang et al., Genes and Dev. (2003)17: 49; Schiebel et al., Plant Cell (1998) 10: 2087 CaenorhabditisSchiebel et al., Plant Cell Elegans (198) 10: 2087 NeurosporaArabidopsis Schiebel et al., Plant Cell (1998) 10: 2087 Drosophiia —Ranjith-Kumar et al., J. HCV Virology (2001) 75: 8615 NS5B Non-specificor specific derived from HCV

A “DNA-dependent RNA polymerase” is an enzyme that synthesizes multipleRNA copies from a double-stranded or partially-double stranded DNAmolecule having a (usually double-stranded) promoter sequence. It shouldbe noted that the present invention includes single stranded promoters,along with the RNA polymerases that recognize them. Examples of DNAdependent RNA polymerases are the DNA-dependent RNA polymerases from E.coli and bacteriophages T7, T3, and SP6. DNA dependant RNA polymerasessuitable for use in the invention are available commercially.

In one embodiment, the DNA dependent RNA polymerase is thermostable.Thermostable RNA polymerases have been derived from Thermus thermophilusis exemplified.

The RNA polymerases can be obtained from either native or recombinantsources. Native viral RNA polymerases, for example, can be isolated fromvirally infected host cells. Examples include infection of HeLa cellswith honey serotype 1 poliovirus using a viral titer and infectionmethodology as described in Pfeiffer et al., Proc. Natl. Acad. Sci.(2003) 100(12):7289-7294. Cellular RNA polymerases can be obtained fromcells that express native RNA polymerases where the cells are grown andthe RNA polymerases harvested as per Schiebel et al (1993). Suitable RNAdependent RNA polymerases are also available commercially.

III. Nucleic Acids

A. Nucleic Acid Sequences Useful in the Invention

The invention provides for methods of detecting or measuring a targetnucleic acid; and also utilizes oligonucleotides, primers and probes forforming a cleavage structure according to the invention and primers foramplifying a template nucleic acid sequence.

As used herein, the terms “nucleic acid”, “polynucleotide” and“oligonucleotide” refer to primers, probes, and oligomer fragments to bedetected, and shall be generic to polydeoxyribonucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and toany other type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, or modified purine or pyrimidine bases (includingabasic sites). There is no intended distinction in length between theterm “nucleic acid”, “polynucleotide” and “oligonucleotide”, and theseterms will be used interchangeably. These terms refer only to theprimary structure of the molecule. Thus, these terms include double- andsingle-stranded DNA, as well as double- and single-stranded RNA.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.”

The oligonucleotide is not necessarily physically derived from anyexisting or natural sequence but may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription ora combination thereof. The terms “oligonucleotide” or “nucleic acid”intend a polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, orsynthetic origin which, by virtue of its synthetic origin ormanipulation: (1) is not associated with all or a portion of thepolynucleotide with which it is associated in nature; and/or (2) islinked to a polynucleotide other than that to which it is linked innature.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of oligonucleotide is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points toward the 5′ end of the other, theformer may be called the “upstream” oligonucleotide and the latter the“downstream” oligonucleotide.

Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids of the present invention and include, forexample, inosine and 7-deazaguanine. Complementarity need not beperfect; stable duplexes may contain mismatched base pairs or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength, andincidence of mismatched base pairs.

Stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m)”. The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which half of the basepairs have disassociated.

B. Primers and Probes Useful According to the Invention

The invention provides for oligonucleotide primers and probes useful fordetecting or measuring a nucleic acid, for amplifying a template nucleicacid sequence, and for forming a cleavage structure according to theinvention.

The term “primer” may refer to more than one primer and refers to anoligonucleotide, whether occurring naturally, as in a purifiedrestriction digest, or produced synthetically, which is capable ofacting as a point of initiation of synthesis along a complementarystrand when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand iscatalyzed. Such conditions include the presence of four differentdeoxyribonucleoside triphosphates and a polymerization-inducing agentsuch as DNA polymerase or reverse transcriptase, in a suitable buffer(“buffer” includes substituents which are cofactors, or which affect pH,ionic strength, etc.), and at a suitable temperature. The primer ispreferably single-stranded for maximum efficiency in amplification.

Oligonucleotide primers useful according to the invention aresingle-stranded DNA or RNA molecules that are hybridizable to a templatenucleic acid sequence and prime enzymatic synthesis of a second nucleicacid strand. The primer is complementary to a portion of a targetmolecule present in a pool of nucleic acid molecules. It is contemplatedthat oligonucleotide primers according to the invention are prepared bysynthetic methods, either chemical or enzymatic. Alternatively, such amolecule or a fragment thereof is naturally-occurring, and is isolatedfrom its natural source or purchased from a commercial supplier.Oligonucleotide primers are 5 to 100 nucleotides in length, ideally from17 to 40 nucleotides, although primers of different length are of use.Primers for amplification are preferably about 17-25 nucleotides.Primers for the production of a cleavage structure according to theinvention are preferably about 17-45 nucleotides. Primers usefulaccording to the invention are also designed to have a particularmelting temperature (Tm) by the method of melting temperatureestimation. Commercial programs, including Oligo™, Primer Design andprograms available on the internet, including Primer3 and OligoCalculator can be used to calculate a Tm of a nucleic acid sequenceuseful according to the invention. Preferably, the Tm of anamplification primer useful according to the invention, as calculatedfor example by Oligo Calculator, is preferably between about 45 and 650C and more preferably between about 50 and 60° C. Preferably, the Tm ofa probe useful according to the invention is 70 C higher than the Tm ofthe corresponding amplification primers.

Primers and probes according to the invention can be labeled and can beused to prepare a labeled cleavage structure. Pairs of single-strandedDNA primers, a DNA primer and a probe or a probe can be annealed tosequences within a target nucleic acid. In certain embodiments, a primercan be used to prime amplifying DNA synthesis of a target nucleic acid.

Typically, selective hybridization occurs when two nucleic acidsequences are substantially complementary (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary). See Kanehisa, M., 1984, Nucleic Acids Res. 12: 203,incorporated herein by reference. As a result, it is expected that acertain degree of mismatch at the priming site is tolerated. Suchmismatch may be small, such as a mono-7, di- or tri-nucleotide.Alternatively, a region of mismatch may encompass loops, which aredefined as regions in which there exists a mismatch in an uninterruptedseries of four or more nucleotides.

Numerous factors influence the efficiency and selectivity ofhybridization of the primer to a second nucleic acid molecule. Thesefactors, which include primer length, nucleotide sequence and/orcomposition, hybridization temperature, buffer composition and potentialfor steric hindrance in the region to which the primer is required tohybridize, will be considered when designing oligonucleotide primersaccording to the invention.

A positive correlation exists between primer length and both theefficiency and accuracy with which a primer will anneal to a targetsequence. In particular, longer sequences have a higher meltingtemperature (T_(M)) than do shorter ones, and are less likely to berepeated within a given target sequence, thereby minimizing promiscuoushybridization. Primer sequences with a high G-C content or that comprisepalindromic sequences tend to self-hybridize, as do their intendedtarget sites, since unimolecular, rather than bimolecular, hybridizationkinetics are generally favored in solution. However, it is alsoimportant to design a primer that contains sufficient numbers of G-Cnucleotide pairings since each G-C pair is bound by three hydrogenbonds, rather than the two that are found when A and T bases pair tobind the target sequence, and therefore forms a tighter, stronger bond.Hybridization temperature varies inversely with primer annealingefficiency, as does the concentration of organic solvents, e.g.formamide, that might be included in a priming reaction or hybridizationmixture, while increases in salt concentration facilitate binding. Understringent annealing conditions, longer hybridization probes, orsynthesis primers, hybridize more efficiently than do shorter ones,which are sufficient under more permissive conditions. Preferably,stringent hybridization is performed in a suitable buffer (for example,1× Sentinel Molecular Beacon PCR core buffer, Stratagene Catalog#600500; 1× Pfu buffer, Stratagene Catalog #200536; or 1× cloned Pfubuffer, Stratagene Catalog #200532) under conditions that allow thenucleic acid sequence to hybridize to the oligonucleotide primers and/orprobes (e.g., 95° C.). Stringent hybridization conditions can vary (forexample from salt concentrations of less than about 1M, more usuallyless than about 500 mM and preferably less than about 200 mM) andhybridization temperatures can range (for example, from as low as 0° C.to greater than 22° C., greater than about 3° C., and (most often) inexcess of about 37° C.) depending upon the lengths and/or the nucleicacid composition or the oligonucleotide primers and/or probes. Longerfragments may require higher hybridization temperatures for specifichybridization. As several factors affect the stringency ofhybridization, the combination of parameters is more important than theabsolute measure of a single factor.

Oligonucleotide primers can be designed with these considerations inmind and synthesized according to the following methods.

1. Oligonucleotide Primer Design Strategy

The design of a particular oligonucleotide primer for the purpose ofsequencing or PCR, involves selecting a sequence that is capable ofrecognizing the target sequence, but has a minimal predicted secondarystructure. The oligonucleotide sequence may or may not bind only to asingle site in the target nucleic acid. Furthermore, the Tm of theoligonucleotide is optimized by analysis of the length and GC content ofthe oligonucleotide. Furthermore, when designing a PCR primer useful forthe amplification of genomic DNA, the selected primer sequence does notdemonstrate significant matches to sequences in the GenBank database (orother available databases).

The design of a primer useful according to the invention, is facilitatedby the use of readily available computer programs, developed to assistin the evaluation of the several parameters described above and theoptimization of primer sequences. Examples of such programs are“PrimerSelect” of the DNAStar™ software package (DNAStar, Inc.; Madison,Wis.), OLIGO 4.0 (National Biosciences, Inc.), PRIMER, OligonucleotideSelection Program, PGEN and Amplify (described in Ausubel et al., 1995,Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons).In one embodiment, primers are designed with sequences that serve astargets for other primers to produce a PCR product that has knownsequences on the ends which serve as targets for further amplification(e.g. to sequence the PCR product). If many different target nucleicacids are amplified with specific primers that share a common ‘tail’sequence’, the PCR products from these distinct genes can subsequentlybe sequenced with a single set of primers. Alternatively, in order tofacilitate subsequent cloning of amplified sequences, primers aredesigned with restriction enzyme site sequences appended to their 5′ends. Thus, all nucleotides of the primers are derived from a targetnucleic acid or sequences adjacent to a target nucleic acid, except forthe few nucleotides necessary to form a restriction enzyme site. Suchenzymes and sites are well known in the art. If the genomic sequence ofa target nucleic acid and the sequence of the open reading frame of atarget nucleic acid are known, design of particular primers is wellwithin the skill of the art.

It is well known by those with skill in the art that oligonucleotidescan be synthesized with certain chemical and/or capture moieties,(including capture elements as defined herein) such that they can becoupled to solid supports and bind to a binding moiety or tag, asdefined herein. Suitable capture elements include, but are not limitedto a nucleic acid binding protein or a nucleotide sequence. Suitablecapture elements include, but are not limited to, biotin, a hapten, aprotein, or a chemically reactive moiety. Such oligonucleotides mayeither be used first in solution, and then captured onto a solidsupport, or first attached to a solid support and then used in adetection reaction. An example of the latter would be to couple adownstream probe molecule to a solid support, such that the 5′ end ofthe downstream probe molecule comprised a fluorescent quencher. The samedownstream probe molecule would also comprise a fluorophore in alocation such that a FEN nuclease cleavage would physically separate thequencher from the fluorophore. For example, the target nucleic acidcould hybridize with the solid-phase downstream probe oligonucleotide,and a liquid phase upstream primer could also hybridize with the targetmolecule, such that a FEN cleavage reaction occurs on the solid supportand liberates the 5′ quencher moiety from the complex. This would causethe solid support-bound fluorophore to be detectable, and thus revealthe presence of a cleavage event upon a suitably labeled or identifiedsolid support. Different downstream probe molecules could be bound to,different locations on an array. The location on the array wouldidentify the probe molecule, and indicate the presence of the templateto which the probe molecule can hybridize.

2. Synthesis

The primers themselves are synthesized using techniques that are alsowell known in the art. Methods for preparing oligonucleotides ofspecific sequence are known in the art, and include, for example,cloning and restriction digest analysis of appropriate sequences anddirect chemical synthesis. Once designed, oligonucleotides are preparedby a suitable chemical synthesis method, including, for example, thephosphotriester method described by Narang et al., 1979, Methods inEnzymology, 68:90, the phosphodiester method disclosed by Brown et al.,1979, Methods in Enzymology, 68:109, the diethylphosphoramidate methoddisclosed in Beaucage et al., 1981, Tetrahedron Letters, 22:1859, andthe solid support method disclosed in U.S. Pat. No. 4,458,066, or byother chemical methods using either a commercial automatedoligonucleotide synthesizer (which is commercially available) or VLSIPS™technology.

C. Probes

The invention provides for probes useful for forming a cleavagestructure or a labeled cleavage structure as defined herein. Methods ofpreparing a labeled cleavage structure according to the invention areprovided in the section entitled “Cleavage Structure” below.

As used herein, the term “probe” refers to a probe that forms a duplexstructure with a sequence in the target nucleic acid, due tocomplementarity of at least one sequence in the probe with a sequence inthe target region. In some embodiments, the probe has a secondarystructure. A probe according to the invention can also be labeled. Theprobe, preferably, does not contain a sequence complementary tosequence(s) used in the primer extension(s). Generally the 3′ terminusof the probe will be “blocked” to prohibit incorporation of the probeinto a primer extension product. Methods of labeling a probe accordingto the invention and suitable labels are described below in the sectionentitled “Cleavage Structure”.

The general design of a probe according to the invention is described inthe section entitled, “Primers and Probes Useful According to theInvention”. Typically, a probe according to the invention comprises atarget nucleic acid binding sequence that is from about 7-140nucleotides, and preferably from about 10-140 nucleotides long (C, FIG.4). In one embodiment, a probe according to the invention also comprisestwo complementary nucleic acid sequence regions, as defined herein (band b′, FIG. 4) that are complementary and bind to each other to form aregion of secondary structure in the absence of a target nucleic acid.Regions b and b′ are 3-25 nucleotides, preferably 4-15 nucleotides andmore preferably 5-11 nucleotides in length. The actual length will bechosen with reference to the target nucleic acid binding sequence suchthat the secondary structure of the probe is preferably stable when theprobe is not bound to the target nucleic acid at the temperature atwhich cleavage of a cleavage structure comprising the probe bound to atarget nucleic acid is performed.

In some embodiments, a probe according to the invention is capable offorming a secondary structure as defined herein, (including a stem loop,a hairpin, an internal loop, a bulge loop, a branched structure and apseudoknot) or multiple secondary structures, cloverleaf type structuresor any three-dimensional structure as defined hereinabove.

For example, according to one embodiment of the present invention, aprobe can be an oligonucleotide with secondary structure such as ahairpin or a stem-loop, and includes, but is not limited to molecularbeacons, safety pins, scorpions, and sunrise/amplifluor probes.

Molecular beacon probes comprise a hairpin, or stem-loop structure whichpossesses a pair of interactive signal generating labeled moieties(e.g., a fluorophore and a quencher) effectively positioned to quenchthe generation of a detectable signal when the beacon probe is nothybridized to the target nucleic acid. The loop comprises a region thatis complementary to a target nucleic acid. The loop is flanked by 5′ and3′ regions (“arms”) that reversibly interact with one another by meansof complementary nucleic acid sequences when the region of the probethat is complementary to a nucleic acid target sequence is not bound tothe target nucleic acid. Alternatively, the loop is flanked by 5′ and 3′regions (“arms”) that reversibly interact with one another by means ofattached members of an affinity pair to form a secondary structure whenthe region of the probe that is complementary to a nucleic acid targetsequence is not bound to the target nucleic acid. As used herein, “arms”refers to regions of a molecular beacon probe that a) reversiblyinteract with one another by means of complementary nucleic acidsequences when the region of the probe that is complementary to anucleic acid target sequence is not bound to the target nucleic acid orb) regions of a probe that reversibly interact with one another by meansof attached members of an affinity pair to form a secondary structurewhen the region of the probe that is complementary to a nucleic acidtarget sequence is not bound to the target nucleic acid. When amolecular beacon probe is not hybridized to target, the arms hybridizewith one another to form a stem hybrid, which is sometimes referred toas the “stem duplex”. This is the closed conformation. When a molecularbeacon probe hybridizes to its target the “arms” of the probe areseparated. This is the open conformation. In the open conformation anarm may also hybridize to the target. Such probes may be free insolution, or they may be tethered to a solid surface. When the arms arehybridized (e.g., form a stem) the quencher is very close to thefluorophore and effectively quenches or suppresses its fluorescence,rendering the probe dark. Such probes are described in U.S. Pat. No.5,925,517 and U.S. Pat. No. 6,037,130.

As used herein, a molecular beacon probe can also be an“allele-discriminating” probe as described herein.

Molecular beacon probes have a fluorophore attached to one arm and aquencher attached to the other arm. The fluorophore and quencher, forexample, tetramethylrhodamine and DABCYL, need not be a FRET pair.

For stem loop probes useful in this invention, the length of the probesequence that is complementary to the target, the length of the regionsof a probe (e.g., stem hybrid) that reversibly interact with one anotherby means of complementary nucleic acid sequences, when the regioncomplementary to a nucleic acid target sequence is not bound to thetarget nucleic acid, and the relation of the two, is designed accordingto the assay conditions for which the probe is to be utilized. Thelengths of the target-complementary sequences and the stem hybridsequences for particular assay conditions can be estimated according towhat is known in the art. The regions of a probe that reversiblyinteract with one another by means of complementary nucleic acidsequences when the region of the probe that is complementary to anucleic acid target sequence is not bound to the target nucleic acid arein the range of 6 to 100, preferably 8 to 50 nucleotides and mostpreferably 8 to 25 nucleotides each. The length of the probe sequencethat is complementary to the target is preferably 17-40 nucleotides,more preferably 17-30 nucleotides and most preferably 17-25 nucleotideslong.

The oligonucleotide sequences of molecular beacon probes modifiedaccording to this invention may be DNA, RNA, cDNA or combinationsthereof. Modified nucleotides may be included, for examplenitropyrole-based nucleotides or 2′-O-methylribonucleotides. Modifiedlinkages also may be included, for example phosphorothioates. Modifiednucleotides and modified linkages may also be incorporated inwavelength-shifting primers according to this invention.

A safety pin probe, as utilized in the present invention, requires a“universal” hairpin probe 1 (FIG. 9, b171 SEQ ID NO: 31), comprising ahairpin structure, with a fluorophore (FAM) on the 5′ arm of the hairpinand a quencher (Dabcyl) on the 3′ arm, and a probe 2 (FIG. 9, SP 170aSEQ ID NO: 32) comprising a stem-loop comprising two domains: the 5′ twothirds of probe 2 (SEQ ID NO: 34) have a (universal) sequencecomplementary to the hairpin probe 1, and nucleotides that will stop theDNA polymerase, and the 3′ one third of probe 2, which serves as thetarget specific primer. As the polymerase, primed from the reverseprimer (that is, the 3′ one third of probe 2) synthesizes the top strand(SEQ ID NO: 33), the 5′ end of probe 2 will be displaced and degraded bythe 5′ exonucleolytic activity until the “stop nucleotides” are reached.At this time the remainder of probe 2 opens up or unfolds and serves asa target for hairpin probe 1 (SEQ ID NO: 31), thereby separating thefluorophore from the quencher (FIG. 9)

Scorpion probes, as used in the present invention comprise a 3′ primerwith a 5′ extended probe tail comprising a hairpin structure whichpossesses a fluorophore/quencher pair. The probe tail is “protected”from replication in the 5′→3′ direction by the inclusion of hexethlyeneglycol (HEG) which blocks the polymerase from replicating the probe.During the first round of amplification the 3′ target-specific primeranneals to the target and is extended such that the scorpion is nowincorporated into the newly synthesized strand, which possesses a newlysynthesized target region for the 5′ probe. During the next round ofdenaturation and annealing, the probe region of the scorpion hairpinloop will hybridize to the target, thus separating the fluorophore andquencher and creating a measurable signal. Such probes are described inWhitcombe et al., Nature Biotechnology 17: 804-807 (1999), and in FIG.10.

An additional oligonucleotide probe useful in the present invention isthe sunrise/amplifluor probe. The sunrise/amplifluor probe is of similarconstruction as the scorpion probe with the exception that is lacks theHEG monomer to block the 5′

3′ replication of the hairpin probe region. Thus, in the first round ofamplification, the 3′ target specific primer of the sunrise/amplifluoranneals to the target and is extended, thus incorporating the hairpinprobe into the newly synthesized strand (sunrise strand). During thesecond round of amplification a second, non-labeled primer anneals tothe 3′ end of the sunrise strand (Cycle 2 in FIG. 11). However, as thepolymerase reaches the 5′ end of the hairpin, due to the lack of the HEGstop sequence, the polymerase will displace and replicate the hairpin,thus separating the fluorophore and quencher, and incorporating thelinearized hairpin probe into the new strand. Probes of this type aredescribed further in Nazarneko et al., Nucleic Acid Res. 25: 2516-2521(1997), and in FIG. 11.

For safety pin, scorpion and sunrise/amplifluor probes useful in thisinvention, the length of the probe sequence that is complementary to thetarget, the length of the regions of a probe (e.g., stem hybrid) thatreversibly interact with one another by means of complementary nucleicacid sequences when the region complementary to a nucleic acid targetsequence is not bound to the target nucleic acid and the relation of thetwo is designed according to the assay conditions for which the probe isto be utilized. The lengths of the target-complementary sequences andthe stem hybrid sequences for particular assay conditions can beestimated according to what is known in the art. The regions of a probethat reversibly interact with one another by means of complementarynucleic acid sequences when the region complementary to a nucleic acidtarget sequence is not bound to the target nucleic acid are in the rangeof 6 to 100, preferably 8 to 50 nucleotides and most preferably 8 to 25nucleotides each. The length of the probe sequence that is complementaryto the target is preferably 17-40 nucleotides, more preferably 17-30nucleotides and most preferably 17-25 nucleotides long. The stability ofthe interaction between the regions of a probe that reversibly interactwith one another by means of complementary nucleic acid sequences isdetermined by routine experimentation to achieve proper functioning. Inaddition to length, the stability of the interaction between the regionsof a probe that reversibly interact with one another by means ofcomplementary nucleic acid sequences can be adjusted by altering the G-Ccontent and inserting destabilizing mismatches. One of the regions of aprobe that reversibly interact with one another by means ofcomplementary nucleic acid sequences can be designed to be partially orcompletely complementary to the target. If the 3′ arm is complementaryto the target the probe can serve as a primer for a DNA polymerase.Also, wavelength-shifting molecular beacon probes can be immobilized tosolid surfaces, as by tethering, or be free-floating.

A wide range of fluorophores may be used in probes and primers accordingto this invention. Available fluorophores include coumarin, fluorescein,tetrachlorofluorescein, hexachlorofluorescein, Lucifer yellow,rhodamine, BODIPY, tetramethylrhodamine, Cy3, Cy5, Cy7, eosine, Texasred and ROX. Combination fluorophores such as fluorescein-rhodaminedimers, described, for example, by Lee et al. (1997), Nucleic AcidsResearch 25:2816, are also suitable. Fluorophores may be chosen toabsorb and emit in the visible spectrum or outside the visible spectrum,such as in the ultraviolet or infrared ranges.

Suitable quenchers described in the art include particularly DABCYL andvariants thereof; such as DABSYL, DABMI and Methyl Red. Fluorophores canalso be used as quenchers, because they tend to quench fluorescence whentouching certain other fluorophores. Preferred quenchers are eitherchromophores such as DABCYL or malachite green, or fluorophores that donot fluoresce in the detection range when the probe is in the openconformation.

D. Production of a Nucleic Acid

The invention provides nucleic acids to be detected and or measured, foramplification of a target nucleic acid and for formation of a cleavagestructure.

The present invention utilizes nucleic acids comprising RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers. The invention includesboth sense and antisense strands of a nucleic acid. According to theinvention, the nucleic acid may be chemically or biochemically modifiedor may contain non-natural or derivatized nucleotide bases. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g. methylphosphonates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators, (e.g. acridine, psoralen, etc.) chelators,alkylators, and modified linkages (e.g. alpha anomeric nucleic acids,etc.) Also included are synthetic molecules that mimic polynucleotidesin their ability to bind to a designated sequence via hydrogen bondingand other chemical interactions. Such molecules are known in the art andinclude, for example, those in which peptide linkages substitute forphosphate linkages in the backbone of the molecule.

1. Nucleic Acids Comprising DNA

a. Cloning

Nucleic acids comprising DNA can be isolated from cDNA or genomiclibraries by cloning methods well known to those skilled in the art(Ausubel et al., supra). Briefly, isolation of a DNA clone comprising aparticular nucleic acid sequence involves screening a recombinant DNA orcDNA library and identifying the clone containing the desired sequence.Cloning will involve the following steps. The clones of a particularlibrary are spread onto plates, transferred to an appropriate substratefor screening, denatured, and probed for the presence of a particularnucleic acid. A description of hybridization conditions, and methods forproducing labeled probes is included below.

The desired clone is preferably identified by hybridization to a nucleicacid probe or by expression of a protein that can be detected by anantibody. Alternatively, the desired clone is identified by polymerasechain amplification of a sequence defined by a particular set of primersaccording to the methods described below.

The selection of an appropriate library involves identifying tissues orcell lines that are an abundant source of the desired sequence.Furthermore, if a nucleic acid of interest contains regulatory sequenceor intronic sequence a genomic library is screened (Ausubel et al.,supra).

b. Genomic DNA

Nucleic acid sequences of the invention are amplified from genomic DNA.Genomic DNA is isolated from tissues or cells according to the followingmethod.

To facilitate detection of a variant form of a gene from a particulartissue, the tissue is isolated free from surrounding normal tissues. Toisolate genomic DNA from mammalian tissue, the tissue is minced andfrozen in liquid nitrogen. Frozen tissue is ground into a fine powderwith a prechilled mortar and pestle, and suspended in digestion buffer(100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, pH 8.0, 0.5% (w/v)SDS, 0.1 mg/ml proteinase K) at 1.2 ml digestion buffer per 100 mg oftissue. To isolate genomic DNA from mammalian tissue culture cells,cells are pelleted by centrifugation for 5 min at 500×g, resuspended in1-10 ml ice-cold PBS, repelleted for 5 min at 500×g and resuspended in 1volume of digestion buffer.

Samples in digestion buffer are incubated (with shaking) for 12-18 hoursat 500 C, and then extracted with an equal volume ofphenol/chloroform/isoamyl alcohol. If the phases are not resolvedfollowing a centrifugation step (10 min at 1700×g), another volume ofdigestion buffer (without proteinase K) is added and the centrifugationstep is repeated. If a thick white material is evident at the interfaceof the two phases, the organic extraction step is repeated. Followingextraction the upper, aqueous layer is transferred to a new tube towhich will be added ½ volume of 7.5M ammonium acetate and 2 volumes of100% ethanol. The nucleic acid is pelleted by centrifugation for 2 minat 1700×g, washed with 70% ethanol, air dried and resuspended in TEbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) at 1 mg/ml. ResidualRNA is removed by incubating the sample for 1 hour at 370 C in thepresence of 0.1% SDS and 1

g/ml DNase-free RNase, and repeating the extraction and ethanolprecipitation steps. The yield of genomic DNA, according to this methodis expected to be approximately 2 mg DNA/1 g cells or tissue (Ausubel etal., supra). Genomic DNA isolated according to this method can be usedfor PCR analysis, according to the invention.

c. Restriction Digest (of cDNA or Genomic DNA)

Following the identification of a desired cDNA or genomic clonecontaining a particular target nucleic acid, nucleic acids of theinvention may be isolated from these clones by digestion withrestriction enzymes.

The technique of restriction enzyme digestion is well known to thoseskilled in the art (Ausubel et al., supra). Reagents useful forrestriction enzyme digestion are readily available from commercialvendors including Stratagene, as well as other sources.

d. PCR

Nucleic acids of the invention may be amplified from genomic DNA orother natural sources by the polymerase chain reaction (PCR). PCRmethods are well-known to those skilled in the art.

PCR provides a method for rapidly amplifying a particular DNA sequenceby using multiple cycles of DNA replication catalyzed by a thermostable,DNA-dependent DNA polymerase to amplify the target sequence of interest.PCR requires the presence of a target nucleic acid to be amplified, twosingle stranded oligonucleotide primers flanking the sequence to beamplified, a DNA polymerase, deoxyribonucleoside triphosphates, a bufferand salts.

PCR, is performed as described in Mullis and Faloona, 1987, MethodsEnzymol., 155: 335, herein incorporated by reference.

The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat.Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is anin vitro method for the enzymatic synthesis of specific DNA sequences,using two oligonucleotide primers that hybridize to opposite strands andflank the region of interest in the target DNA. A repetitive series ofreaction steps involving template denaturation, primer annealing and theextension of the annealed primers by DNA polymerase results in theexponential accumulation of a specific fragment whose termini aredefined by the 5′ ends of the primers. PCR is reported to be capable ofproducing a selective enrichment of a specific DNA sequence by a factorof 10⁹. The PCR method is also described in Saiki et al., 1985, Science230:1350.

PCR is performed using template DNA (at least 1 fg; more usefully,1-1000 ng) and at least 25 pmol of oligonucleotide primers. A typicalreaction mixture includes: 2 μl of DNA, 25 pmol of oligonucleotideprimer, 2.5 μl of a suitable buffer, 0.4 μl of 1.25 μM dNTP, 2.5 unitsof Taq DNA polymerase (Stratagene) and deionized water to a total volumeof 25 μl. Mineral oil is overlaid and the PCR is performed using aprogrammable thermal cycler.

The length and temperature of each step of a PCR cycle, as well as thenumber of cycles, are adjusted according to the stringency requirementsin effect. Annealing temperature and timing are determined both by theefficiency with which a primer is expected to anneal to a template andthe degree of mismatch that is to be tolerated. The ability to optimizethe stringency of primer annealing conditions is well within theknowledge of one of moderate skill in the art. An annealing temperatureof between 30° C. and 72° C. is used. Initial denaturation of thetemplate molecules normally occurs at between 92° C. and 99° C. for 4minutes, followed by 20-40 cycles consisting of denaturation (94-99° C.for 15 seconds to 1 minute), annealing (temperature determined asdiscussed above; 1-2 minutes), and extension (72° C. for 1 minute). Thefinal extension step is generally carried out for 4 minutes at 72° C.,and may be followed by an indefinite (0-24 hour) step at 4° C.

Detection methods generally employed in standard PCR techniques use alabeled probe with the amplified DNA in a hybridization assay.Preferably, the probe is labeled, e.g., with ³²P, biotin, horseradishperoxidase (HRP), etc., to allow for detection of hybridization.

Other means of detection include the use of fragment length polymorphism(PCR FLP), hybridization to allele-specific oligonucleotide (ASO) probes(Saiki et al., 1986, Nature 324:163), or direct sequencing via thedideoxy method (using amplified DNA rather than cloned DNA). Thestandard PCR technique operates (essentially) by replicating a DNAsequence positioned between two primers, providing as the major productof the reaction a DNA sequence of discrete length terminating with theprimer at the 5′ end of each strand. Thus, insertions and deletionsbetween the primers result in product sequences of different lengths,which can be detected by sizing the product in PCR-FLP. In an example ofASO hybridization, the amplified DNA is fixed to a nylon filter (by, forexample, UV irradiation) in a series of “dot blots”, then allowed tohybridize with an oligonucleotide probe labeled with HRP under stringentconditions. After washing, terramethylbenzidine (TMB) and hydrogenperoxide are added: HRP oxidizes the hydrogen peroxide, which in turnoxidizes the TMB to a blue precipitate, indicating a hybridized probe.

A PCR assay for detecting or measuring a nucleic assay according to theinvention is described in the section entitled “Methods of Use”.

2. Nucleic Acids Comprising RNA

The present invention also provides a nucleic acid comprising RNA.

Nucleic acids comprising RNA can be purified according to methods wellknown in the art (Ausubel et al., supra). Total RNA can be isolated fromcells and tissues according to methods well known in the art (Ausubel etal., supra) and described below.

RNA is purified from mammalian tissue according to the following method.Following removal of the tissue of interest, pieces of tissue of ≦2 gare cut and quick frozen in liquid nitrogen, to prevent degradation ofRNA. Upon the addition of a suitable volume of guanidinium solution (forexample 20 ml guanidinium solution per 2 g of tissue), tissue samplesare ground in a tissuemizer with two or three 10-second bursts. Toprepare tissue guanidinium solution (1 L) 590.8 g guanidiniumisothiocyanate is dissolved in approximately 400 ml DEPC-treated H₂O. 25ml of 2 M Tris-HCl, pH 7.5 (0.05 M final) and 20 ml Na₂EDTA (0.01 Mfinal) is added, the solution is stirred overnight, the volume isadjusted to 950 ml, and 50 ml 2-ME is added.

Homogenized tissue samples are subjected to centrifugation for 10 min at12,000×g at 120 C. The resulting supernatant is incubated for 2 min at650 C in the presence of 0.1 volume of 20% Sarkosyl, layered over 9 mlof a 5.7M CsCl solution (0.1 g CsCl/ml), and separated by centrifugationovernight at 113,000×g at 220 C. After careful removal of thesupernatant, the tube is inverted and drained. The bottom of the tube(containing the RNA pellet) is placed in a 50 ml plastic tube andincubated overnight (or longer) at 40 C in the presence of 3 ml tissueresuspension buffer (5 mM EDTA, 0.5% (v/v) Sarkosyl, 5% (v/v) 2-ME) toallow complete resuspension of the RNA pellet. The resulting RNAsolution is extracted sequentially with 25:24:1phenol/chloroform/isoamyl alcohol, followed by 24:1 chloroform/isoamylalcohol, precipitated by the addition of 3 M sodium acetate, pH 5.2, and2.5 volumes of 100% ethanol, and resuspended in DEPC water (Chirgwin etal., 1979, Biochemistry, 18: 5294).

Alternatively, RNA is isolated from mammalian tissue according to thefollowing single step protocol. The tissue of interest is prepared byhomogenization in a glass teflon homogenizer in 1 ml denaturing solution(4M guanidinium thiosulfate, 25 mM sodium citrate, pH 7.0, 0.1M 2-ME,0.5% (w/v) N-laurylsarkosine) per 100 mg tissue. Following transfer ofthe homogenate to a 5-ml polypropylene tube, 0.1 ml of 2 M sodiumacetate, pH 4, 1 ml water-saturated phenol, and 0.2 ml of 49:1chloroform/isoamyl alcohol are added sequentially. The sample is mixedafter the addition of each component, and incubated for 15 min at 0-40 Cafter all components have been added. The sample is separated bycentrifugation for 20 min at 10,000×g, 4° C., precipitated by theaddition of 1 ml of 100% isopropanol, incubated for 30 minutes at −20°C. and pelleted by centrifugation for 10 minutes at 10,000×g, 4° C. Theresulting RNA pellet is dissolved in 0.3 ml denaturing solution,transferred to a microfuge tube, precipitated by the addition of 0.3 mlof 100% isopropanol for 30 minutes at −20° C., and centrifuged for 10minutes at 10,000×g at 4° C. The RNA pellet is washed in 70% ethanol,dried, and resuspended in 100-200 μl DEPC-treated water or DEPC-treated0.5% SDS (Chomczynski and Sacchi, 1987, Anal. Biochem., 162: 156).

Nucleic acids comprising RNA can be produced according to the method ofin vitro transcription.

The technique of in vitro transcription is well known to those of skillin the art. Briefly, the gene of interest is inserted into a vectorcontaining an SP6, T3 or T7 promoter. The vector is linearized with anappropriate restriction enzyme that digests the vector at a single sitelocated downstream of the coding sequence. Following a phenol/chloroformextraction, the DNA is ethanol precipitated, washed in 70% ethanol,dried and resuspended in sterile water. The in vitro transcriptionreaction is performed by incubating the linearized DNA withtranscription buffer (200 mM Tris-HCl, pH 8.0, 40 mM MgCl₂, 10 mMspermidine, 250 NaCl [T7 or T3] or 200 mM Tris-HCl, pH 7.5, 30 mM MgCl₂,10 mM spermidine [SP6]), dithiothreitol, RNase inhibitors, each of thefour ribonucleoside triphosphates, and either SP6, T7 or T3 RNApolymerase for 30 min at 370 C. To prepare a radiolabeled polynucleotidecomprising RNA, unlabeled UTP will be omitted and ³⁵S-UTP will beincluded in the reaction mixture. The DNA template is then removed byincubation with DNaseI. Following ethanol precipitation, an aliquot ofthe radiolabeled RNA is counted in a scintillation counter to determinethe cpm/

1 (Ausubel et al., supra).

Alternatively, nucleic acids comprising RNA are prepared by chemicalsynthesis techniques such as solid phase phosphoramidite (describedabove).

3. Nucleic Acids Comprising Oligonucleotides

A nucleic acid comprising oligonucleotides can be made by usingoligonucleotide

synthesizing machines which are commercially available (describedabove).

IV. Cleavage Structure

The invention provides for a cleavage structure that can be cleaved by anuclease (e.g., a FEN nuclease), and therefore teaches methods ofpreparing a cleavage structure.

The invention also provides a labeled cleavage structure and methods ofpreparing a labeled cleavage structure.

A probe is used to prepare a cleavage structure according to theinvention.

A. Preparation of a Cleavage Structure

In one embodiment, a cleavage structure according to the invention isformed by incubating a) an upstream oligonucleotide primer, b) anoligonucleotide probe located not more than 5000 nucleotides downstreamof the upstream primer and c) an appropriate target nucleic acid whereinthe target sequence is complementary to both primer and probe and d) asuitable buffer (for example 10× Pfu buffer available from Stratagene(Catalog #200536) or buffers compatible with the particular RNApolymerase used, under conditions that allow the nucleic acid sequenceto hybridize to the oligonucleotide primers (for example 95° C. for 1-2minutes followed by cooling to between approximately 40-72° C.). Theoptimal temperature will vary depending on the specific probe(s),primers and RNA polymerase. In some embodiments of the invention, acleavage structure comprises an overlapping flap wherein the 3′ end ofan upstream oligonucleotide capable of hybridizing to a target nucleicacid (for example A in FIG. 4) is complementary to 1 or more basepair(s) of the downstream oligonucleotide probe (for example C in FIG.4) that is annealed to a target nucleic acid.

In another embodiment, a cleavage structure according to the inventionis formed by incubating a) an RNA polymerase synthesizedoligonucleotide, b) an oligonucleotide probe located not more than 5000nucleotides downstream of a promoter and c) an appropriate targetnucleic acid wherein the target sequence comprises a promoter region andis at least partially complementary to downstream probe, and d) asuitable buffer under conditions that allow the nucleic acid sequence tohybridize to the oligonucleotide primers (for example 95° C. for 1-2minutes followed by cooling to between approximately 40-60° C.). Theoptimal temperature will vary depending on the specific probe(s) and RNApolymerase. In some embodiments of the invention, a cleavage structurecomprises an overlapping flap wherein the 3′ end of an upstreamoligonucleotide capable of hybridizing to a target nucleic acid (forexample A in FIG. 4) is complementary to 1 or more base pair(s) of thedownstream oligonucleotide probe (for example C in FIG. 4) that isannealed to a target nucleic acid.

In one embodiment, a cleavage structure according to the invention isformed by incubating a) an upstream, preferably extendable 3′ end,preferably an oligonucleotide primer, b) an oligonucleotide probe havinga secondary structure, as defined herein, that changes upon binding to atarget nucleic acid and comprising a binding moiety, located not morethan 5000 nucleotides downstream of the upstream primer and c) anappropriate target nucleic acid wherein the target sequence iscomplementary to both primers and d) a suitable buffer (for exampleSentinel Molecular Beacon PCR-core buffer (Catalog #600500) or 10× Pfubuffer available from Stratagene (Catalog #200536), under conditionsthat allow the nucleic acid sequence to hybridize to the oligonucleotideprimers (for example 95° C. for 2-5 minutes followed by cooling tobetween approximately 50-60° C.). The optimal temperature will varydepending on the specific probe(s), primers and polymerases. Inpreferred embodiments of the invention a cleavage structure comprises anoverlapping flap wherein the 3′ end of an upstream oligonucleotidecapable of hybridizing to a target nucleic acid (for example A in FIG.4) is complementary to 1 or more base pair(s) of the downstreamoligonucleotide probe (for example C in FIG. 4) that is annealed to atarget nucleic acid and wherein the 1 base pair overlap is directlydownstream of the point of extension of the single stranded flap.

According to this embodiment of the 3′ end of the upstreamoligonucleotide primer is extended by the synthetic activity of apolymerase according to the invention such that the newly synthesized 3′end of the upstream oligonucleotide primer partially displaces the 5′end of the downstream oligonucleotide probe. Extension is preferablycarried out in the presence of 1× Sentinel Molecular beacon core bufferor 1× Pfu buffer for 15 seconds at 72° C.

In another embodiment of the invention, a cleavage structure accordingto the invention can be prepared by incubating a target nucleic acidwith a partially complementary oligonucleotide probe having a secondarystructure, as defined herein, that changes upon binding to a targetnucleic acid and comprising a binding moiety, to a target nucleic acidsuch that the 3′ complementary region anneals to the target nucleic acidand the non-complementary 5′ region that does not anneal to the targetnucleic acid forms a 5′ flap. Annealing is preferably carried out underconditions that allow the nucleic acid sequence to hybridize to theoligonucleotide primer (for example 95° C. for 2-5 minutes followed bycooling to between approximately 50-60° C.) in the presence a suitablebuffer (for example 1× Sentinel Molecular beacon core buffer or 1× Pfubuffer.

In another embodiment of the invention, a cleavage structure accordingto the invention can be prepared by incubating a target nucleic acidwith an upstream primer capable of hybridizing to the target nucleicacid and a partially complementary oligonucleotide probe having asecondary structure, as defined herein, that changes upon binding to atarget nucleic acid and comprising a binding moiety, such that the 3′complementary region anneals to the target nucleic acid and thenon-complementary 5′ region that does not anneal to the targetnucleic-acid forms a 5′ flap. Annealing is preferably carried out underconditions that allow the nucleic acid sequence to hybridize to theoligonucleotide primer (for example 95 C for 2-5 minutes followed bycooling to between approximately 50-60° C.) in the presence a suitablebuffer (for example 1× Sentinel Molecular beacon core buffer(Stratagene) or 1×Pfu buffer (Stratagene).

B. How to Prepare a Labeled Cleavage Structure

In the present invention, a label is attached to an oligonucleotideprobe. In one embodiment of the present invention, a label is attachedto an oligonucleotide probe having a secondary structure, as definedherein, that changes upon binding to a target nucleic acid andcomprising a binding moiety, that comprises a cleavage structure. Thus,the cleaved mononucleotides or small oligonucleotides which are cleavedby the endonuclease activity of the flap-specific nuclease can bedetected.

In one embodiment, a labeled cleavage structure according to theinvention is formed by incubating a) an upstream extendable 3′ end,preferably an oligonucleotide primer, b) a labeled probe having asecondary structure, as defined herein, that changes upon binding to atarget nucleic acid and comprising a binding moiety, located not morethan 5000 nucleotides downstream of the upstream primer and c) anappropriate target nucleic acid wherein the target sequence iscomplementary to the oligonucleotides and d) a suitable buffer (forexample 1× Sentinel Molecular beacon core buffer or 1× Pfu buffer),under conditions that allow the nucleic acid sequence to hybridize tothe oligonucleotide primers (for example 95° C. for 2-5 minutes followedby cooling to between approximately 50-60° C.). A cleavage structureaccording to the invention also comprises an overlapping flap whereinthe 3′ end of an upstream oligonucleotide capable of hybridizing to atarget nucleic acid (for example A in FIG. 4) is complementary to 1 basepair of the downstream oligonucleotide probe having a secondarystructure, as defined herein, that changes upon binding to a targetnucleic acid comprising a binding moiety (for example C in FIG. 4) thatis annealed to a target nucleic acid and wherein the 1 base pair overlapis directly downstream of the point of extension of the single strandedflap. The 3′ end of the upstream primer is extended by the syntheticactivity of a polymerase such that the newly synthesized 3′ end of theupstream primer partially displaces the labeled 5′ end of the downstreamprobe. Extension is preferably carried out in the presence of 1×Sentinel Molecular beacon core buffer or 1× Pfu buffer for 15 seconds at72° C.

In another embodiment, a cleavage structure according to the inventioncan be prepared by incubating a target nucleic acid with a probe havinga secondary structure, as defined herein, that changes upon binding to atarget nucleic acid and comprising a binding moiety, and furthercomprising a non-complementary, labeled, 5′ region that does not annealto the target nucleic acid and forms a 5′ flap, and a complementary 3′region that anneals to the target nucleic acid. Annealing is preferablycarried out under conditions that allow the nucleic acid sequence tohybridize to the oligonucleotide primer (for example 95° C. for 2-5minutes followed by cooling to between approximately 50-60° C.) in thepresence a suitable buffer (for example 1× Sentinel Molecular beaconcore buffer or 1× Pfu buffer).

In another embodiment, a cleavage structure according to the inventioncan be prepared by incubating a target nucleic acid with an upstreamprimer that is capable of hybridizing to the target nucleic acid and aprobe having a secondary structure, as defined herein, that changes uponbinding to a target nucleic acid and comprising a binding moiety, andfurther comprising a non-complementary, labeled, 5′ region that does notanneal to the target nucleic acid and forms a 5′ flap; and acomplementary 3′ region that anneals to the target nucleic acid.Annealing is preferably carried out under conditions that allow thenucleic acid sequence to hybridize to the oligonucleotide primer (forexample 95° C. for 2-5 minutes followed by cooling to betweenapproximately 50-60° C.) in the presence a suitable buffer (for example1× Sentinel Molecular beacon core buffer or 1× Pfu-buffer).

Subsequently, any of several strategies may be employed to distinguishthe uncleaved labeled nucleic acid from the cleaved fragments thereof.The invention provides for methods for detecting the amount of cleaved,released, nucleic acid fragment that is captured by binding of a bindingmoiety or a tag to a capture element, on a solid support. In thismanner, the present invention permits identification of those samplesthat contain a target nucleic acid.

The oligonucleotide probe is labeled, as described below, byincorporating moieties detectable by spectroscopic, photochemical,biochemical, immunochemical, enzymatic or chemical means. The method oflinking or conjugating the label to the oligonucleotide probe depends,of course, on the type of label(s) used and the position of the label onthe probe. A probe that is useful according to the invention can belabeled at the 5′ end, the 3′ end or labeled throughout the length ofthe probe.

A variety of labels that would be appropriate for use in the invention,as well as methods for their inclusion in the probe, are known in theart and include, but are not limited to, enzymes (e.g., alkalinephosphatase and horseradish peroxidase) and enzyme substrates,radioactive atoms, fluorescent dyes, chromophores, chemiluminescentlabels, electrochemiluminescent labels, such as Origen™ (Igen), that mayinteract with each other to enhance, alter, or diminish a signal. Ofcourse, if a labeled molecule is used in a PCR based assay carried outusing a thermal cycler instrument, the label must be able to survive thetemperature cycling required in this automated process.

Among radioactive atoms, ³³P or, ³²P is preferred. Methods forintroducing ³³P or, ³²P into nucleic acids are known in the art, andinclude, for example, 5′ labeling with a kinase, or random insertion bynick translation. “Specific binding partner” refers to a protein capableof binding a ligand molecule with high specificity, as for example inthe case of an antigen and a monoclonal antibody specific therefor Otherspecific binding partners include biotin and avidin or streptavidin, IgGand protein A, and the numerous receptor-ligand couples known in theart. The above description is not meant to categorize the various labelsinto distinct classes, as the same label may serve in several differentmodes. For example, ¹²⁵I may serve as a radioactive label or as anelectron-dense reagent. HRP may serve as an enzyme or as antigen for amonoclonal antibody. Further, one may combine various labels for desiredeffect. For example, one might label a probe with biotin, and detect thepresence of the probe with avidin labeled with ¹²⁵I, or with ananti-biotin monoclonal antibody labeled with HRP. Other permutations andpossibilities will be readily apparent to those of ordinary skill in theart and are considered as equivalents within the scope of the instantinvention.

Fluorophores for use as labels in constructing labeled probes of theinvention include rhodamine and derivatives (such as Texas Red),fluorescein and derivatives (such as 5-bromomethyl fluorescein), LuciferYellow, IAEDANS, 7-Me₂N-coumarin-4-acetate,7-OH-4-CH₃-coumarin-3-acetate, 7—NH₂-4-CH₃-coumarin-3-acetate (AMCA),monobromobimane, pyrene trisulfonates, such as Cascade Blue, andmonobromorimethyl-ammoniobimane. In general, fluorophores with wideStokes shifts are preferred, to allow using fluorimeters with filtersrather than a monochromometer and to increase the efficiency ofdetection.

Probes labeled with fluorophores can readily be used in nuclease (e.g.FEN-nuclease) mediated cleavage of a cleavage structure comprising alabeled probe according to the invention. If the label is on the 5′-endof the probe, the nuclease (e.g. FEN-nuclease) generated labeledfragment is separated from the intact, hybridized probe by procedureswell known in the art.

In another embodiment of the invention, detection of the hydrolyzed,labeled probe can be accomplished using, for example, fluorescencepolarization, a technique to differentiate between large and smallmolecules based on molecular tumbling. Large molecules (i.e., intactlabeled probe) tumble in solution much more slowly than small molecules.Upon linkage of a fluorescent moiety to an appropriate site on themolecule of interest, this fluorescent moiety can be measured (anddifferentiated) based on molecular tumbling, thus differentiatingbetween intact and digested probe.

In some situations, one can use two interactive labels (e.g., FRET ornon-FRET pairs) on a single oligonucleotide probe with due considerationgiven for maintaining an appropriate spacing of the labels on theoligonucleotide to permit the separation of the labels duringoligonucleotide probe unfolding (e.g., for example due to a change inthe secondary structure of the probe) or hydrolysis. Preferredinteractive labels useful according to the invention include, but arenot limited to rhodamine and derivatives, fluorescein and derivatives,Texas Red, coumarin and derivatives, crystal violet and include, but arenot limited to DABCYL, TAMRA and NTB (nitrothiazole blue) in addition toany of the FRET or non-FRET labels described herein.

The fluorescence of the released label is then compared to the labelremaining bound to the target. It is not necessary to separate thenuclease (e.g. FEN-nuclease) generated fragment and the probe thatremains bound to the target after cleavage in the presence of nuclease(e.g. FEN-nuclease) if the probe is synthesized with a fluorophore and aquencher that are separated by about 20 nucleotides. Alternatively, thequencher is positioned such that the probe will not fluoresce when nothybridized to the target nucleic acid. Such a dual labeled probe willnot fluoresce when intact or when not hybridized to the target nucleicacid (or in the case of bi- or multimolecular probes, when the probe isnot dissociated) because the light emitted from the dye is quenched bythe quencher. Thus, any fluorescence emitted by an intact probe isconsidered to be background fluorescence. In one embodiment, when alabeled probe is cleaved by a FEN nuclease, dye and quencher areseparated and the released fragment will fluoresce. Alternatively, whena labeled probe is hybridized to a target nucleic acid, the distancebetween the dye and the quencher is increased and the level offluorescence increases. In an embodiment wherein the probe is a bi- ormulti-molecular probe, dissociation of the molecules comprising theprobe results in an increase in fluorescence. The amount of fluorescenceis proportional to the amount of nucleic acid target sequence present ina sample.

In yet another embodiment, two labeled nucleic acids are used, eachcomplementary to separate regions of separate strands of adouble-stranded target sequence, but not to each other, so that alabeled nucleic acid anneals downstream of each primer. For example, thepresence of two probes can potentially double the intensity of thesignal generated from a single label and may further serve to reduceproduct strand reannealing, as often occurs during PCR amplification.The probes are selected so that the probes bind at positions adjacent(downstream) to the positions at which primers bind.

One can also use multiple probes in the present invention to achieveother benefits. For instance, one could test for any number of pathogensin a sample simply by putting as many probes as desired into thereaction mixture; the probes could each comprise a different label tofacilitate detection.

One can also achieve allele-specific or species-specific discriminationusing multiple probes in the present invention, for instance, by usingprobes that have different T_(m)s and conducting the annealing/cleavagereaction at a temperature specific for only one probe/allele duplex. Onecan also achieve allele specific discrimination by using only a singleprobe and examining the types of cleavage products generated. In oneembodiment of the invention, the probe is designed to be exactlycomplementary, at least in the 5′ terminal region, to one allele but notto the other allele(s). With respect to the other allele(s), the probewill be mismatched in the 5′ terminal region of the probe so that adifferent cleavage product will be generated as compared to the cleavageproduct generated when the probe is hybridized to the exactlycomplementary allele.

Although probe sequence can be selected to achieve important benefits,one can also realize important advantages by selection of probelabels(s) and/or tag as defined herein. The labels may be attached tothe oligonucleotide directly or indirectly by a variety of techniques.Depending on the precise type of label or tag used, the label can belocated at the 5′ or 3′ end of the probe, located internally in theprobe, or attached to spacer arms of various sizes and compositions tofacilitate signal interactions. Using commercially availablephosphoramidite reagents, one can produce oligomers containingfunctional groups (e.g., thiols or primary amines) at either the 5- orthe 3-terminus via an appropriately protected phosphoramidite, and canlabel them using protocols described in, for example, PCR Protocols: AGuide to Methods and Applications, Innis et al., eds. Academic Press,Ind., 1990.

Methods for introducing oligonucleotide functionalizing reagents tointroduce one or more sulfhydryl, amino or hydroxyl moieties into theoligonucleotide probe sequence, typically at the 5′ terminus, aredescribed in U.S. Pat. No. 4,914,210. A 5′ phosphate group can beintroduced as a radioisotope by using polynucleotide kinase andgamma-³²P-ATP or gamma-³³P-ATP to provide a reporter group. Biotin canbe added to the 5′ end by reacting an aminothymidine residue, or a6-amino hexyl residue, introduced during synthesis, with anN-hydroxysuccinimide ester of biotin. Labels at the 3′ terminus mayemploy polynucleotide terminal transferase to add the desired moiety,such as for example, cordycepin ³⁵S-dATP, and biotinylated dUTP.

Oligonucleotide derivatives are also available labels. For example,etheno-dA and etheno-A are known fluorescent adenine nucleotides thatcan be incorporated into a nucleic acid probe. Similarly, etheno-dC or2-amino purine deoxyriboside is another analog that could be used inprobe synthesis. The probes containing such nucleotide derivatives maybe hydrolyzed to release much more strongly fluorescent mononucleotidesby flap-specific nuclease activity.

C. Cleaving a Cleavage Structure and Generating a Signal

A cleavage structure according to the invention can be cleaved by themethods described in the section above, entitled “Nucleases”.

D. Detection of Released Labeled Fragments

Detection or verification of the labeled fragments may be accomplishedby a variety of methods well known in the art and may be dependent onthe characteristics of the labeled moiety or moieties comprising alabeled cleavage structure.

In one embodiment of the invention, the reaction products, including thereleased labeled fragments; are subjected to size analysis. Methods fordetermining the size of a labeled fragment are known in the art andinclude, for example, gel electrophoresis, sedimentation in gradients,gel exclusion chromatography, mass spectroscopy, and homochromatography.In another embodiment, a detectable signal is generated upon theseparation of a pair of interactive labels.

In one embodiment, the released labeled fragments are captured bybinding of a binding moiety to a capture element attached to a solidsupport.

1. Capture Element

A capture element, according to the invention can be any moiety thatspecifically binds (e.g. via hydrogen bonding or via an interactionbetween, for example a nucleic acid binding protein and a nucleic acidbinding site or between complementary nucleic acids) a binding moiety,as a result of attractive forces that exist between the binding moietyand the capture element.

According to the invention, a binding moiety includes a region of aprobe that binds to a capture element. A capture element according tothe invention can be a nucleic acid sequence that is complementary toand binds to, for example, via hydrogen bonding, a binding moietycomprising a region of a probe that binds to a capture element. Forexample, a binding moiety is a region of a probe comprising the nucleicacid sequence 5′AGCTACTGATGCAGTCACGT3′ (SEQ ID NO: 26) and thecorresponding capture element comprises the nucleic acid sequence5′TCGATGACTACGTCAGTGCA3′ (SEQ ID NO: 27).

The invention also provides for binding moiety-capture element ortag-capture element pairs wherein the binding moiety or tag is a DNAbinding protein and the corresponding capture element is the DNAsequence recognized and bound by the DNA binding protein. The inventionalso provides for binding moiety-capture element or tag-capture elementpairs wherein the capture element is a DNA binding protein and thecorresponding binding moiety or tag is the DNA sequence recognized andbound by the DNA binding protein.

DNA sequence/DNA binding protein interactions useful according to theinvention include but are not limited to c-myb, AAF, abd-A, Abd-B,ABF-2, ABF1, ACE2, ACF, ADA2, ADA3, Adf-1, Adf-2a, ADR1, AEF-1, AF-2,AFP1, AGIE-BP1, AhR, AIC3, AIC4, AID2, AIIN3, ALFIB, alpha-1, alpha-CP1,alpha-CP2a, alpha-CP2b, alpha-factor, alpha-PAL, alpha2uNF1, alpha2uNF3,alphaA-CRYBP1, alphaH2-alphaH3, alphaMHCBF1, aMEF-2, AML1, AnCF, ANF,ANF-2, Antp, AP-1, AP-2, AP-3, AP-5, APETALA1, APETALA3, AR, ARG RI, ARGRII, Arnt, AS-C T3, AS321, ASF-1, ASH-1, ASH-3b, ASP, AT-13P2, ATBF1-A,ATF, ATF-1, ATF-3, ATF-3deltaZIP, ATF-adelta, ATF-like, Athb-1, Athb-2,Axial, abaA, ABF-1, Ac, ADA-NF1, ADD1, Adf-2b, AF-1, AG, AIC2, AIC5,ALF1A, alpha-CBF, alpha-CP2a, alpha-CP2b, alpha-IRP, alpha2uNF2,alphaH0, AmdR, AMT1, ANF-1, Ap, AP-3, AP-4, APETALA2, aRA, ARG RIII,ARP-1, Ase, ASH-3a, AT-BP1, ATBF1-B, ATF-2, ATF-a, ATF/CREB, Ato, Bfactor, B″, B-Myc, B-TFIID, band I factor, BAP, Bcd, BCFI, Bcl-3,beta-1, BETA1, BETA2, BF-1, BGP1, BmFTZ-F1, BP1, BR-C Z1, BR-C Z2, BR-CZ4, Brachyury, BRF1, BrlA, Bm-3a, Bm-4, Bm-5, BUF1, BUF2, B-Myb, BAF1,BAS1, BCFII, beta-factor, BETA3, BLyF, BP2, BR-C Z3, brahma, byr3,c-abl, c-Ets-1, c-Ets-2, c-Fos, c-Jun, c-Maf, c-myb, c-Myc, c-Qin,c-Rel, C/EBP, C/EBPalpha, C/EBPbeta, C/EBPdelta, C/EBPepsilon,C/EBPgamma, C1, CAC-binding protein, CACCC-binding factor, Cactus, Cad,CAD1, CAP, CArG box-binding protein, CAUP, CBF, CBP, CBTF, CCAAT-bindingfactor, CCBF, CCF, CCK-1a, CCK-1b, CD28RC, CDC10, Cdc68, CDF, cdk2, CDP,Cdx-1, Cdx-2, Cdx-3, CEBF, CEH-18, CeMyoD, CF1, Cf1a, CF2-I, CF2-II,CF2-III, CFF, CG-1, CHOP-10, Chox-2.7, CIIIB1, Clox, Cnc, CoMP1,core-binding factor, CoS, COUP, COUP-TF, CP1, CP1A, CP1B, CP2, CPBP,CPC1, CPE binding protein CPRF-1, CPRF-2, CPRF-3, CRE-BP1, CRE-BP2,CRE-BP3, CRE-BPa, CreA, CREB, CREB-2, CREBomega, CREMalpha, CREMbeta,CREMdelta, CREMepsilon, CREMgamma, CREMtaualpha, CRF, CSBP-1, CTCF, CTF,CUP2, Cut, Cux, Cx, cyclin A, CYS3, D-MEF2, Da, DAL82, DAP, DAT1, DBF-A,DBF4, DBP, DBSF, dCREB, dDP, dE2F, DEF, Delilah, delta factor,deltaCREB, deltaE1, deltaEF1, deltaMax, DENF, DEP, DF-1, Dfd, dFRA,dioxin receptor, dJRA, D1, DII, D1x, DM-SSRP1, DMLP1, DP-1, Dpn, Dr1,DRTF, DSC1; DSP1, DSXF, DSXM, DTF, E, E1A, E2, E2BP, E2F, E2F-BF, E2F-I,E4, E47, E4BP4, E4F, E4TF2, E7, E74, E75, EBF, EBF1, EBNA, EBP, EBP40,EC, ECF, ECH, EcR, eE-TF, EF-1A, EF-C, EF1, EFgamma, Egr, eH-TF, EIIa,EivF, EKLF, Elf-1, Elg, Elk-1, ELP, Elt-2, EmBP-1, embryo DNA bindingprotein, Emc, EMF, Ems, Emx, En, ENH-binding protein, ENKTF-1,epsilonF1, ER, Erg, Esc, ETF, Eve, Evi, Evx, Exd, Ey, f(alpha-epsilon),F-ACT1, f-EBP, F2F, factor 1-3, factor B1, factor B2, factor delta,factor I, FBF-A1, Fbf1, FKBP59, Fkh, F1bD, Flh, Fli-1, FLV-1, Fos-B,Fra-2, FraI, FRG Y1, FRG Y2, FTS, Ftz, Ftz-F1, G factor, G6 factor,GA-BF, GABP, GADD 153, GAF, GAGA factor, GAL4, GAL80, gamma-factor,gammaCAAT, gammaCAC, gammaOBP, GATA-1, GATA-2, GATA-3, GBF, GC1, GCF,GCF, GCN4, GCR1, GE1, GEBF-I, GF1, GFI, Gfi-1, GFII, GHF-5, GLi, Glass,GLO, GM-PBP-1, GP, GR, GRF-1, Gsb, Gsbn, Gsc, Gt, GT-1, Gtx, H, H16,H1lTF1, H₂Babp1, H2RIIBP, H2TF1, H4TF-1, HAC1, HAP1, Hb, HBLF, HBP-1,HCM1, heat-induced factor, HEB, HEF-1B, HEF-1T, HEF-4C, HEN1, HES-1,HIF-1, HiNF-A, HIP1, HIV-EP2, Hlf, HMBI, HNF-1, HNF-3, Hox11, HOXA1,HOXA10, HOXA10PL2, HOXA11, HOXA2, HOXA3, HOXA4, HOXA5, HOXA7, HOXA9,HOXB1, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXC5, HOXC6,HOXC8, HOXD1, HOXD10, HOXD11, HOXD12, HOXD13, HOXD4, HOXD8, HOXD9, HP1site factor, Hp55, Hp65, HrpF, HSE-binding protein, HSF1, HSF2, HSF24,HSF3, HSF30, HSF8, hsp56, Hsp90, HST, HSTF, I—POU, IBF, IBP-1, ICER,ICP4, ICSBP, Id1, Id2, Id3, Id4, IE1, EBP1, IEFga, IF1, IF2, IFNEX,IgPE-1, IK-1, IkappaB, Il-1 RF, IL-6 RE-BP, Il-6 RF, ILF, ILRF-A, IME1,INO2, INSAF, IPF1, IRBP, IRE-ABP, IREBF-1, IRF-1, ISGF-1, Isl-1, ISRF,ITF, IUF-1, Ixr1, JRF, Jun-D, JunB, JunD, K-2, kappay factor, kBF-A,KBF1, KBF2, KBP-1, KER-1, Ker1, KN1, Kni, Knox31 Kr, kreisler, KRF-1,Krox-20, Krox-24, Ku autoantigen, KUP, Lab, LAC9, LBP, Lc, LCR-F1,LEF-1, LEF-1S, LEU3, LF-A1, LF-B1, LF-C, LF-H3beta, LH-2, Lim-1, Lim-3,lin-11, lin-31, lin-32, LIP, LIT-1, LKLF, Lmx-1, LRF-1, LSF, LSIRF-2,LVa, LVb-binding factor, LVc, LyF-1, Lyl-1, M factor, M-Twist, M1, m3,Mab-18, MACI1 Mad, MAF, MafB, MafF, MafG, MafK, Ma163, MAPF1, MAPF2,MASH-1, MASH-2, mat-Mc, mat-Pc, MATa1, MATalpha1, MATalpha2, MATH-1,MATH-2, Max1, MAZ, MBF-1, MBP-1, MBP-2, MCBF, MCM1, MDBP, MEB-1, Mec-3,MECA, mediating factor, MEF-2, MEF-2C, MEF-2D, MEF1, MEP-1, Mesol, MF3,Mi, MIF, MIG1, MLP, MNB1a, MNF1, MOK-2, MP4, MPBF, MR, MRF4, MSN2, MSN4,Msx-1, Msx-2, MTF-1, mtTF1, muEBP-B, muEBP-C2, MUF1, MUF2, Mxi1, Myef-2,Myf-3, Myf-4, Myf-5, Myf-6, Myn, MyoD, myogenin, MZF-1, N-Myc, N-Oct-2,N-Oct-3, N-Oct-4, N-Oct-5, Nau, NBF, NC1, NeP1, Net, NeuroD, neurogenin,NF III-a, NF-1, NF-4FA, NF-AT, NF-BA1, NF-CLE0a, NF-D, NF-E, NF-E1b,NF-E2, NF-EM5, NF-GMa, NF-H1, NF-IL-2A, NF-InsE1, NF-kappaB, NF-lambda2,NF-MHCIIA, NF-muE1, NF-muNR, NF-S, NF-TNF, NF-U1, NF-Wi, NF-X, NF-Y,NF-Zc, NFalphal, NFAT-1, NFbetaA, NFdeltaE3A, NFdeltaE4A, NFe, NFE-6,NFH3-1, NFH3-2, NFH3-3, NFH3-4, NGFI-B, NGFI-C, NHP, Nil-2-a, NIP, NIT2,Nkx-2.5, NLS1, NMH7, NP-III, NP-IV, NP-TCII, NP-Va, NRDI, NRF-1, NRF-2,Nrf1, Nrf2, NRL, NRSF form 1, NTF, NUC-1, Nur77, OBF, OBP, OCA-B, OCSTF,Oct-1, Oct-10, Oct-11, Oct-2, Oct-2.1, Oct-2.3, Oct-4, Oct-5, Oct-6,Oct-7, Oct-8, Oct-9, Oct-B2, Oct-R, Octa-factor, octamer-binding factor,Odd, O1f-1, Opaque-2, Otd, Otx1, Otx2, Ovo, P, P1, p107, p130, p28modulator, p300, p38erg, p40x, p45, p49erg, p53, p55, p55erg, p58,p65delta, p67, PAB1, PacC, Pap1, Paraxis, Pax-1, Pax-2, Pax-3, Pax-5,Pax-6, Pax-7, Pax-8, Pb, Pbx-1a, Pbx-1b, PC, PC2, PC4, PC5, Pcr1, PCRE1,PCT1, PDM-1, PDM-2, PEA1, PEB1, PEBP2, PEBP5, Pep-1, PF1, PGA4, PHD1,PHO2, PHO4, PHO80, Phox-2, Pit-1, PO-B, pointedP1, Pou2, PPAR, PPUR,PPYR, PR, PR A, Prd, PrDI-BF1, PREB, Prh proein a, protein b, proteinc,protein d, PRP, PSE1, PTF, Pu box binding factor, PU.1, PUB1, PuF,PUF-I, Pur factor, PUT3, pX, qa-1F, QBP, R, R1, R2, RAd-1, RAF, RAP1,RAR, Rb, RBP-Jkappa, RBP60, RC1, RC2, REB1, RelA, RelB, repressor ofCAR1 expression, REX-1, RF-Y, RF1, RFX, RGM1, RIM1, RLM1, RME1, Ro,RORalpha, Rox1, RPF1, RPGalpha, RREB-1, RRF1, RSRFC4, runt, RVF,RXR-alpha, RXR-beta, RXR-beta2, RXR-gamma, S-CREM, S-CREMbeta, S8,SAP-1a, SAP1, SBF, Sc, SCBPalpha, SCD1/BP, SCM-inducible factor, Scr,Sd, Sdc-1, SEF-1, SF-1, SF-2, SF-3, SF-A, SGC1, SGF-1, SGF-2, SGF-3,SGF-4, SIF, SIII, Sim, SIN1, Skn-1, SKO1, Slp1, Sn, SNP1, SNF5, SNAPC43,Sox-18, Sox-2, Sox-4, Sox-5, Sox-9, Sox-LZ, Sp1, spE2F, Sph factor,Spi-B, Sprm-1, SRB10, SREBP, SRF, SRY, SSDBP-1, ssDBP-2, SSRP1, STAF-50,STAT, STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, STC, STD1, Ste11, Ste12,Step4, STM, Su(f), SUM-1, SWI1, SWI4, SWI5, SWI6, SWP, T-Ag, t-Pou2,T3R, TAB, all TAFs including subunits, Tal-1, TAR factor, tat, Tax,TBF1, TBP, TCF, TDEF, TEA1, TEC1, TEF, tel, Tf-LF1, TFE3, all TFIIrelated proteins, TBA1a, TGGCA-binding protein, TGT3, Th1, TIF1, TIN-1,TIP, T11, TMF, TR2, Tra-1, TRAP, TREB-1, TREB-2, TREB-3, TREF1, TREF2,Tsh, TTF-1, TTF-2, Ttk69k, TTP, Ttx, TUBF, Twi, TxREBP, TyBF, UBP-1,Ubx, UCRB, UCRF-L, UF1-H3beta, UFA, UFB, UHF-1, UME6, Unc-86, URF, URSF,URTF, USF, USF2, v-ErbA, v-Ets, v-Fos, v-Jun, v-Maf, v-Myb, v-Myc,v-Qin, v-Rel, Vab-3, vaccinia virus DNA-binding protein, Vav, VBP, VDR,VETF, vHNF-1, VITF, Vmw65, Vp1, Vp16, Whn, WT1, X-box binding protein,X-Twist, X2BP, XBP-1, XBP-2, XBP-3, XF1, XF2, XFD-1, XFD-3, xMEF-2,XPF-1, XrpFI, XW, XX, yan, YB-1, YEB3, YEBP, Yi, YPF1, YY1, ZAP, ZEM1,ZEM2/3, Zen-1, Zen-2, Zeste, ZF1, ZF2, Zfh-1, Zfh-2, Zfp-35, ZID,Zmhoxla, Zta and all related characterized and uncharacterized homologsand family members related to these DNA binding proteins or activities,and the DNA sequence recognized by the above-recited DNA bindingproteins. Methods of identifying a DNA sequence recognized by a DNAbinding protein are known in the art (see for example, U.S. Pat. No.6,139,833).

The invention also contemplates DNA sequence/DNA binding proteininteractions including but not limited to the tetracycline (tet)repressor, beta.-galactosidase (lac repressor), the tryptophan (trp)repressor, the lambda specific repressor protein, CRO, and thecatabolite activator-protein, CAP and the DNA sequence recognized byeach of these DNA binding proteins and known in the art. DNA/DNA bindingprotein interactions usefull according to the invention also includerestriction enzymes and the corresponding restriction sites, preferablyunder conditions wherein the nuclease activity of the restriction enzymeis suppressed (U.S. Pat. No. 5,985,550, incorporated herein byreference).

Other DNA:Protein interactions useful according to the invention include(i) the DNA protein interactions listed in Tables 1 and 2, and (ii)bacterial, yeast, and phage systems such as lambda OL-OR/CrO (U.S. Pat.No. 5,726,014, incorporated herein by reference). Any pair comprising aprotein that binds to a specific recognition sequence and the cognaterecognition sequence may be useful in the present invention.

TABLE 1 DNA-BINDING SEQUENCES DNA-binding Test sequence Protein EBVorigin of replication EBNA HSV origin of replication UL9 VZV origin ofreplication UL9-like HPV origin of replication E2 Interleukin 2 enhancerNFAT-1 HIV LTR NFAT-1 NFkB HBV enhancer HNF-1 Fibrogen promoter HNF-1

TABLE 2 Name DNA Sequence Recognized* Bacteria lac AATTGTGAGCGGATAACAATTrepressor (SEQ ID NO: 4) TTAACACTCGCCTATTGTTAA (SEQ ID NO: 5) CAPTGTGAGTTAGCTCACT (SEQ ID NO: 6) ACACTCAATCGAGTGA (SEQ ID NO: 7) lambdaTATCACCGCCAGAGGTA repressor (SEQ ID NO: 8) ATAGTGGCGGTCTCCAT (SEQ ID NO:9) Yeast GAL4 CGGAGGACTGTCCTCCG (SEQ ID NO: 10) GCCTCCTGACAGGAGGC (SEQID NO: 11) MAT ˜2 CATGTAATT (SEQ ID NO: 12) GTACATTAA (SEQ ID NO: 13)GCN4 ATGACTCAT (SEQ ID NO: 14) TACTGAGTA (SEQ ID NO: 15) DrosophilaKrüppel AACGGGTTAA (SEQ ID NO: 16) TTGCCCAATT (SEQ ID NO: 17) bicoidGGGATTAGA (SEQ ID NO: 18) CCCTAATCT (SEQ ID NO: 19) Mammals Sp1 GGGCGG(SEQ ID NO: 20) CCCGCC (SEQ ID NO: 21) Oct-1 ATGCAAAT (SEQ ID NO: 22)TACGTTTA (SEQ ID NO: 23) GATA-1 TGATAG (SEQ ID NO: 24) ACTATC (SEQ IDNO: 25) *Each protein in this table can recognize a set of closelyrelated DNA sequences; for convenience, only one recognition sequence isgiven for each protein.

Methods of performing a reaction wherein specific binding occurs betweena capture element, as defined herein and a binding moiety, as definedherein, are well known in the art, see for example, Sambrook et al.,supra; Ausubel et al., supra). A capture element, according to theinvention can also be any moiety that specifically binds (e.g. viacovalent or hydrogen bonding or electrostatic attraction or via aninteraction between, for example a protein and a ligand, an antibody andan antigen, protein subunits, a nucleic acid binding protein and anucleic acid binding site) a binding moiety or a tag, as a result ofattractive forces that exist between the binding moiety or tag and thecapture element. Methods of performing a reaction wherein specificbinding occurs between a capture element, as defined herein and a tag,as defined herein, are well known in the art, see for example, Sambrooket al., supra; Ausubel et al., supra). Specific binding only occurs whenthe secondary structure of the probe comprising the binding moiety has“changed”, as defined herein. Capture elements useful according to theinvention include but are not limited to a nucleic acid binding proteinor a nucleotide sequence, biotin, streptavidin, a hapten, a protein, anucleotide sequence or a chemically reactive moiety.

In one embodiment of the invention, the reaction products, including thereleased labeled fragments, are subjected to size analysis. Methods fordetermining the size of a labeled fragment are known in the art andinclude, for example, gel electrophoresis, sedimentation in gradients,gel exclusion chromatography, mass spectroscopy, and homochromatography.

2. Solid Substrate

A solid substrate according to the invention is any surface to which amolecule (e.g., capture element) can be irreversibly bound, includingbut not limited to membranes, magnetic beads, tissue culture plates,silica based matrices, membrane based matrices, beads comprisingsurfaces including but not limited to styrene, latex or silica basedmaterials and other polymers for example cellulose acetate, teflon,polyvinylidene difluoride, nylon, nitrocellulose, polyester, carbonate,polysulphone, metals, zeolites, paper, alumina, glass, polypropyle,polyvinyl chloride, polyvinylidene chloride, polytetrafluorethylene,polyethylene, polyamides, plastic, filter paper, dextran, germanium,silicon, (poly)tetrafluorethylene, gallium arsenide, gallium phosphide,silicon oxide, silicon nitrate and combinations thereof.

Useful solid substrates according to the invention are also described inSambrook et al., supra, Ausubel et al., supra, U.S. Pat. Nos. 5,427,779,5,512,439, 5,589,586, 5,716,854 and 6,087,102, Southern et al., 1999,Nature Genetics Supplement, 21:5 and Joos et al., 1997, AnalyticalBiochemistry, 247:96.

Methods of attaching a capture element to a solid support are known inthe art and are described in Sambrook et al., supra, Ausubel et al.,supra, U.S. Pat. Nos. 5,427,779, 5,512,439, 5,589,586, 5,716,854 and6,087,102 and in Southern et al., supra and Joos et al., supra. Methodsof immobilizing a nucleic acid sequence on a solid support are alsoprovided by the manufacturers of the solid support, e.g., for membranes:Pall Corporation, Schleicher & Schuell, for magnetic beads; Dyal, forculture plates; Costar, Nalgenunc, and for other supports usefulaccording to the invention, CPG, Inc.

The amount of released labeled fragment that is bound to a captureelement attached to a solid support can be measured while the labeledfragment remains bound to the capture element or after release of thelabeled fragment from the capture element. Release of a labeled fragmentfrom a capture element is carried out by incubating labeledfragment-capture element complexes in the presence of an excess amountof a competing, unlabeled fragment or by the addition of a buffer thatinhibits binding of the labeled fragment to the capture element, forexample as a result of salt concentration or pH.

During or after amplification, separation of the released labeledfragments from, for example, a PCR mixture can be accomplished by, forexample, contacting the PCR with a solid phase extractant (SPE). Forexample, materials having an ability to bind nucleic acids on the basisof size, charge, or interaction with the nucleic acid bases can be addedto the PCR mixture, under conditions where labeled, uncleaved nucleicacids are bound and short, labeled fragments are not. Such SPE materialsinclude ion exchange resins or beads, such as the commercially availablebinding particles Nensorb (DuPont Chemical Co.), Nucleogen (The NestGroup), PEI, BakerBond™ PEI, Amicon PAE 1,000, Selectacel™ PEI, BoronateSPE with a 3′-ribose probe, SPE containing sequences complementary tothe 3′-end of the probe, and hydroxylapatite. In a specific embodiment,if a dual labeled oligonucleotide comprising a 3′ biotin label separatedfrom a 5′ label by a nuclease susceptible cleavage site is employed asthe signal means, the reaction mixture, for example a PCR amplifiedmixture can be contacted with materials containing a specific bindingelement such as avidin or streptavidin, or an antibody or monoclonalantibody to biotin, bound to a solid support such as beads andparticles, including magnetic particles.

Following the step in which a reaction mixture, for example a PCRmixture has been contacted with an SPE, the SPE material can be removedby filtration, sedimentation, or magnetic attraction, leaving thelabeled fragments free of uncleaved labeled oligonucleotides andavailable for detection.

3. Binding Moieties

A binding moiety according to the invention refers to a region of aprobe that is released upon cleavage of the probe by a nuclease andbinds specifically (via hydrogen binding with a complementary nucleicacid or via an interaction with a binding protein) to a capture elementas a result of attractive forces that exist between the binding moietyand the capture element, and wherein specific binding between thebinding moiety and the capture element only occurs when the secondarystructure of the probe has “changed”, as defined herein.

A “tag” refers to a moiety that is operatively linked to the 5′ end of aprobe (for example R in FIG. 1) and specifically binds to a captureelement as a result of attractive forces that exist between the tag andthe capture element, and wherein specific binding between the tag andthe capture element only occurs when the secondary structure of theprobe has changed (for example, such that the tag is accessible to acapture element). “Specifically binds” as it refers to a “tag” and acapture element means via covalent or hydrogen bonding or electrostaticattraction or via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, or a nucleic acidbinding protein and a nucleic acid binding site. Second binding moietiesinclude but are not limited to biotin, streptavidin, a hapten; aprotein, or a chemically reactive moiety.

According to the invention, a binding moiety includes a region of aprobe that binds to a capture element. A capture element according tothe invention can be a nucleic acid sequence that is complementary toand binds to, for example, via hydrogen bonding, a binding moietycomprising a region of a probe that binds to a capture element. Forexample, a binding moiety is a region of a probe comprising the nucleicacid sequence 5′AGCTACTGATGCAGTCACGT3′ (SEQ ID NO: 26) and thecorresponding capture element comprises the nucleic acid sequence5′TCGATGACTACGTCAGTGCA3′ (SEQ ID NO: 27).

The invention also provides for binding moiety-capture element ortag-capture element pairs wherein the binding moiety or tag is a DNAbinding protein and the corresponding capture element is the DNAsequence recognized and bound by the DNA binding protein. The inventionalso provides for binding moiety-capture element or tag-capture elementpairs wherein the capture element is a DNA binding protein and thecorresponding binding moiety or tag is the DNA sequence recognized andbound by the DNA binding protein.

DNA binding sequence/DNA binding protein interactions useful accordingto the invention are described above in the section entitled, “Detectionof Released Labeled Fragments”.

Methods of incorporating a tag, as defined herein, into a nucleic acid(e.g., a probe according to the invention) are well known in the art andare described in Ausubel et al., supra, Sambrook et al., supra, and U.S.Pat. Nos. 5,716,854 and 6,087,102.

IV. Determining the Stability of the Secondary Structure of a Probe

A. Melting Temperature Assay

A melting temperature assay, takes advantage of the different absorptionproperties of double stranded and single stranded DNA, that is, doublestranded DNA (the double stranded DNA being that portion of a nucleicacid sequence that has folded back on itself to generate an antiparallelduplex structure wherein complementary sequences (base pairs) areassociated via hydrogen bonding) absorbs less light than single strandedDNA at a wavelength of 260 nm, as determined by spectrophotometricmeasurement.

The denaturation of DNA occurs over a narrow temperature range andresults in striking changes in many of the physical properties of DNA. Aparticularly useful change occurs in optical density. The heterocyclicrings of nucleotides adsorb light strongly in the ultraviolet range(with a maximum close to 260 nm that is characteristic for each base).However, the adsorption of DNA is approximately 40% less than would bedisplayed by a mixture of free nucleotides of the same composition. Thiseffect is called hyperchromism and results from interactions between theelectron systems of the bases, made possible by their stacking in theparallel array of the double helix. Any departure from the duplex stateis immediately reflected by a decline in this effect (that is, by anincrease in optical density toward the value characteristic of freebases (FIG. 12 a). The denaturation of double stranded DNA can thereforebe followed by this hyperchromicity (FIGS. 12 b and 12 c)

The Midpoint of the Temperature Range Over which the Strands of DNASeparate is called the melting temperature, denoted T_(m). An example ofa melting curve determined by change in optical absorbance is shown inFIG. 12 c. The curve always takes the same form, but its absoluteposition on the temperature scale (that is, its T_(m)) is influenced byboth the base composition of the DNA and the conditions employed fordenaturation.

The melting temperature of a DNA molecule depends markedly on its basecomposition. DNA molecules rich in GC base pairs have a higher Tm thanthose having an abundance of AT base pairs (FIG. 13 b). The Tm of DNAfrom many species varies linearly with GC content, rising from 77° to100° C. as the fraction of GC pairs increases from 20% to 78%. That is,the dependence of T_(m) on base composition is linear, increasing about0.4° C. for every percent increase in G-C content. GC base pairs aremore stable than AT pairs because their bases are held together by threehydrogen bonds rather than by two. In addition, adjacent GC base pairsinteract more strongly with one another than do adjacent AT base pairs.Hence, the AT-rich regions of DNA are the first to melt.

A major effect on T_(m) is exerted by the ionic strength of thesolution. The T_(m) increases 16.6° C. for every tenfold increase inmonovalent cation concentration. The most commonly used condition is toperform manipulations of DNA in 0.12 M phosphate buffer, which providesa monovalent Na+ concentration of 0.18M, and a T_(m) of the order of 90°C.

The T_(m) can be greatly varied by performing the reaction in thepresence of reagents, such as form amide, that destabilize hydrogenbonds. This allows the T_(m) to be reduced to as low as 40° C. with theadvantage that the DNA does not suffer damage (such as strand breakage)that can result from exposure to high temperatures. (Stryer,Biochemistry, 1998, 3^(rd) Edition, W.H. Freeman and Co., pp. 81-82 andLewin, Genes II, 1985, John Wiley & Sons, p. 63-64).

The stability of the secondary structure of the probe according to theinvention is determined in a melting temperature assay as follows.

A standard curve for the probe (for example FIG. 12 c), whereinabsorbance is plotted versus temperature, is prepared by incubating asample comprising from about 0.2 μg/ml to 100 μg/ml of the probe in abuffer which allows for denaturing and reannealing of the probe atvarious temperatures for a time sufficient to permit denaturing andreannealing of the probe and measuring the absorbance of a sample in aquartz cuvette (with a pathlength appropriate for the spectrophotometerbeing used, e.g., 1-cm), in a spectrophotometer over a range oftemperatures wherein the lower temperature limit of the range is atleast 50° C. below, and the upper temperature limit of the range is atleast 50° C. above the Tm or predicted Tm of the probe. The Tm of theprobe is predicted based on the base pair composition according tomethods well known in the art (see, Sambrook, supra; Ausubel, supra).Standard curves are generated and compared, using a variety of buffers(e.g., 1× TNE buffer (10×-0.1M Tris base, 10 mM EDTA, 2.0 M NaCl, pH7.4), FEN nuclease buffer, described herein, 1× Cloned Pfu buffer;described herein, 1× Sentinel Molecular beacon buffer, described herein)including a buffer that is possible and preferentially optimal for theparticular nuclease to be employed in the cleavage reaction. The pH ofthe buffer will be monitored as the temperature increases, and adjustedas is needed.

The assay is performed in a single-beam ultraviolet to visible range(UV-VIS) spectrophotometer. Preferably, the assay is performed in adouble-beam spectrophotometer to simplify measurements by automaticallycomparing the cuvette holding the sample solution to a reference cuvette(matched cuvette) that contains the blank. The blank is an equal volumeof sample buffer.

The temperature of the spectrophotometer can be controlled such that theabsorbance of the sample is measured at specific temperatures.Spectrophotometers useful according to the invention include but are notlimited to the Beckman Coulter DU® 600/7000 Spectrophotometers incombination with the Micro™ Analysis Accessory (Beckman Coulter, Inc.,Columbia, Md.).

The stability of the secondary structure of a probe at a particulartemperature and in a buffer that is possible and preferentially optimalfor the nuclease to be employed in the cleavage reaction of the probe,is determined by measuring the absorbance of the probe at a particulartemperature, as above, and determining if the value of the absorbance isless than the absorbance at the Tm, as determined from the standardcurve, wherein the standard curve is generated using either the samebuffer as used at the test temperature, or a buffer known to produce acomparable standard curve, as described above. The secondary structureof the probe is “stable” in a melting temperature assay, at atemperature that is at or below the temperature of the cleavage reaction(i.e., at which cleavage is performed) if the level of light absorbanceat the temperature at or below the temperature of the cleavage reactionis less (i.e., at least 5%, preferably 20% and most preferably 25% ormore) than the level of light absorbance at a temperature that is equalto the Tm of the probe (see FIGS. 12 c and 12 d).

B. FRET

A FRET assay is useful in the invention for two purposes. The first isto determine whether the secondary structure of a probe is “stable” asdefined herein. The second is to determine whether the secondarystructure of the probe has undergone a “change” upon binding of theprobe to the target nucleic acid. “FRET” is a distance dependentinteraction between the electronic excited states of two dye moleculesin which excitation is transferred from a donor molecule to an acceptormolecule. FRET is caused by a change in the distance separating afluorescent donor group from an interacting resonance energy acceptor,either another fluorophore, a chromophore, or a quencher. Combinationsof donor and acceptor moieties are known as “FRET pairs”. Efficient FRETinteractions require that the absorption and emission spectra of the dyepairs have a high degree of overlap.

In most embodiments, the donor and acceptor dyes for FRET are different,in which case FRET can be detected by the appearance of sensitizedfluorescence of the acceptor and/or by quenching of donor fluorescence.When the donor and acceptor are the same, FRET is detected by theresulting fluorescence depolarization. FRET is dependent on the inversesixth power of the intermolecular separation (Stryer et al., 1978, Ann.Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300).

As used herein, the term “donor” refers to a fluorophore which absorbsat a first wavelength and emits at a second, longer wavelength. The term“acceptor” refers to a fluorophore, chromophore or quencher with anabsorption spectrum which overlaps the donor's emission spectrum and isable to absorb some or most of the emitted energy from the donor when itis near the donor group (typically between 1-100 nm). If the acceptor isa fluorophore capable of exhibiting FRET, it then re-emits at a third,still longer wavelength; if it is a chromophore or quencher, then itreleases the energy absorbed from the donor without emitting a photonAlthough the acceptor's absorption spectrum overlaps the donor'semission spectrum when the two groups are in proximity, this need not bethe case for the spectra of the molecules when free in solution.Acceptors thus include fluorophores, chromophores or quenchers whichexhibit either FRET or quenching when placed in proximity, on a probeaccording to the invention, to the donor due to the presence of a probesecondary structure that changes upon binding of the probe to the targetnucleic acid, as defined herein. Acceptors do not include fluorophores,chromophores or quenchers that exhibit FRET or quenching a) attemperatures equal to or greater than the Tm (e.g. more than 5° abovethe Tm, for example 6°, 10°, 25°, 50° or more above the Tm) or b) in thepresence of a target nucleic acid.

Reference herein to “fluorescence” or “fluorescent groups” or“fluorophores” include luminescence, luminescent groups and suitablechromophores, respectively. Suitable luminescent probes include, but arenot limited to, the luminescent ions of europium and terbium introducedas lanthium chelates (Heyduk & Heyduk, 1997). The lanthanide ions arealso good donors for energy transfer to fluorescent groups (Selvin1995). Luminescent groups containing lanthanide ions can be incorporatedinto nucleic acids utilizing an ‘open cage’ chelator phosphoramidite.

As used herein, the term “quenching” refers to the transfer of energyfrom donor to acceptor which is associated with a reduction of theintensity of the fluorescence exhibited by the donor.

The donor and acceptor groups may independently be selected fromsuitable fluorescent groups, chromophores and quenching groups. Donorsand acceptors useful according to the invention include but are notlimited to: 5-FAM (also called 5-carboxyfluorescein; also calledSpiro(isobenzofuran-1(3H), 9′-(9H)xanthene)-5-carboxylicacid,3′,6′-dihydroxy-3-oxo-6-carboxyfluorescein);5-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-6-carboxylicacid]); 6-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 5-Tetrachloro-Fluorescein([4,7,2′,7′-tetra-chloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 6-Tetrachloro-Fluorescein([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-6-6-carboxylicacid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium,9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA(6-carboxytetramethylrhodamine; Xanthylium,9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS(5-((((2-iodoacetyl)amino)ethyl) amino)naphthalene-1-sulfonic acid);DABCYL (4-((4-(dimethylamino)phenyl) azo)benzoic acid) Cy5(Indodicarbocyanine-5) Cy3 (Indo-dicarbocyanine-3); and BODIPY FL(2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionicacid), as well as suitable derivatives thereof.

In certain embodiments of the invention, a probe may also be labeledwith two chromophores, and a change in the absorption spectra of thelabel pair is used as a detection signal, as an alternative to measuringa change in fluorescence.

In the method of the invention, fluorescence intensity of the probe ismeasured at one or more wavelengths with a fluorescencespectrophotometer or microtitre plate reader, according to methods knownin the art.

C. Fluorescence Quenching Assay

A fluorescence quenching assay is useful in the invention for twopurposes. The first is to determine whether the secondary structure of aprobe is “stable” as defined herein. The second is to determine whetherthe secondary structure of the probe has undergone a “change” uponbinding of the probe to the target nucleic acid.

A probe according to the invention is labeled with a pair of interactivelabels (e.g., a FRET or non-FRET pair) wherein one member of the pair isa fluorophore and the other member of the pair is a quencher. Forexample, a probe according to the invention is labeled with afluorophore and a quencher and fluorescence is measured in the absenceof a target nucleic acid, over a range of temperatures, e.g., whereinthe lower temperature limit of the range is at least 50° Celsius below,and the upper temperature limit of the range is at least 50° Celsiusabove the Tm or the predicted Tm of the probe.

D. Stability

The “stability” of the secondary structure of a probe according to theinvention is determined as follows. A probe is labeled with a pair ofinteractive labels (for example, tetramethylrhodamine and DABCYL, or anyof the interactive labels (either FRET or non-FRET pairs) describedherein according to methods well known in the art (for example asdescribed in Glazer and Mathies, 1997, Curr. Opin. Biotechnol., 8:94; Juet al., 1995, Analytical Biochem., 231:131)). The location of theinteractive labels on the probe is such that the labels are separatedwhen the secondary structure of the probe changes following binding ofthe probe to the target nucleic acid.

A standard curve for the probe (for example FIG. 12 e), whereinfluorescence is plotted versus temperature, is prepared by incubating asample comprising typically 125 nM probe in 1× Melting Buffer (20 mMTris-HCl, pH 8.0, 1 mM MgCl₂) or alternatively, in 5 mM Tris-HCl, pH8.0, 0.1 mM EDTA, or other appropriate buffers for a time that issufficient to permit denaturing and reannealing of the probe (typicallythe standard curve is generated using a fluorometer or spectrometer thatundergoes a 1° C. per minute change, and measuring the fluorescence in afluorometer or scanning fluorescence spectrophotometer over a range oftemperatures wherein the lower temperature limit of the range is atleast 50° C. below, and the upper temperature limit of the range is atleast 50° C. above the Tm or predicted Tm of the probe. The Tm of theprobe is predicted based on the base pair composition according tomethods well known in the art (see, Sambrook, supra; Ausubel, supra).

Standard curves are generated and compared, using a variety of buffers(e.g., 1× TNE buffer (10×-0.1M Tris base, 10 mM EDTA, 2.0 M NaCl, pH7.4), FEN nuclease buffer, described herein, 1× Cloned Pfu buffer,described herein, 1× Sentinel Molecular beacon buffer, described herein)including a buffer that is possible and preferentially optimal for theparticular nuclease to be employed in the cleavage reaction. The pH ofthe buffer will be monitored as the temperature increases, and adjustedas is needed.

The temperature of the fluorometer or spectrophotometer can becontrolled such that the fluorescence of the sample is measured atspecific temperatures. Fluorescence can be measured for example with aPerkin-Elmer LS50B Luminescence Spectrometer in combination with atemperature regulatable water bath (e.g., for example available fromFisher Scientific).

The stability of the secondary structure of a probe at a particulartemperature is determined by measuring the fluorescence of the probe ata particular temperature, as above, and determining if the value of thefluorescence is less than the fluorescence at the Tm, as determined fromthe standard curve. The secondary structure of the probe is “stable” ina FRET assay, at a temperature that is at or below the temperature ofthe cleavage reaction (i.e., at which cleavage is performed) if thelevel of fluorescence at the temperature at or below the temperature ofthe cleavage reaction is altered (i.e., at least 5%, preferably 20% andmost preferably 25% more or less than) the level of fluorescence at atemperature that is equal to the Tm of the probe. The secondarystructure of the probe is “stable” in a fluorescence quenching assay, ata temperature that is at or below the temperature of the cleavagereaction (i.e., at which cleavage is performed) if the level offluorescence at the temperature at or below the temperature of thecleavage reaction is less (i.e., at least 5%, preferably 20% and mostpreferably 25% more or less than) the level of fluorescence at atemperature that is equal to the Tm of the probe (see FIGS. 12 f and 12g).

Alternatively, the stability of the secondary structure of the probe isdetermined by modifying the method of Gelfand et al. (1999, Proc. Natl.Acad. Sci. USA, 96:6113), incorporated herein by reference, to determinethe fluorescence of a probe labeled with a pair of interactive labelsover a range of temperatures, as described hereinabove.

V. Detecting a Secondary Structure

A secondary structure according to the invention is detected bygenerating a standard curve of fluorescence versus temperature for aprobe comprising a pair of interactive labels in a FRET or fluorescencequenching assay, as described above (see FIG. 12 e). A probe thatexhibits a change in fluorescence that correlates with a change intemperature (see FIG. 12 e) (e.g., fluorescence increases as thetemperature of the FRET reaction is increased) is capable of forming asecondary structure.

VI. Measuring a Change in Secondary Structure

A “change” in secondary structure according to the invention is detectedby analyzing a probe comprising a pair of interactive labels in a FRETor fluorescence quenching assay at a particular temperature below the Tmof the probe, (e.g., the cleaving temperature), as described above, inthe presence or absence of 100 nM to 10 μM of a target nucleic acid(typically the target nucleic acid is in a 2-4 molar excess over theprobe concentration, i.e., 250-500 nM target nucleic acid is used).

Alternatively, a change in the secondary structure of the probe isdetermined by modifying the method of Gelfand et al. (1999, Proc. Natl.Acad. Sci. USA, 96:6113), incorporated herein by reference, to determinethe fluorescence of a probe labeled with a pair of interactive labels inthe presence or absence of a target nucleic acid as describedhereinabove.

A “change” in secondary structure that occurs when a probe according tothe invention binds to a target nucleic acid, is measured as an increasein fluorescence, such that the level of fluorescence after binding ofthe probe to the target nucleic acid at the temperature below the Tm ofthe probe, is greater than (e.g., at least 5%, preferably 5-20% and morepreferably 25 or more) the level of fluorescence observed in the absenceof a target nucleic acid (see FIG. 12 g).

VII. Methods of Use

The invention provides for a method of generating a signal indicative ofthe presence of a target nucleic acid in a sample comprising the stepsof forming a labeled cleavage structure by incubating a target nucleicacid with a probe and cleaving the cleavage structure with a nuclease(e.g. a FEN nuclease). The method of the invention can be used in a PCRbased assay as described below.

A labeled cleavage structure comprising an upstream oligonucleotideprimer (for example A, FIG. 4), a 5′ end labeled downstreamoligonucleotide probe and a target nucleic acid (for example B in FIG.4) is formed as described above in the section entitled “CleavageStructure”. In some embodiments, the downstream probe has a secondarystructure that changes upon binding to the target nucleic acid andcomprises a binding moiety (for example C in FIG. 4). Briefly, acleavage structure is formed and cleaved in the presence of a targetnucleic acid, in the presence or absence of an upstream primer (forexample A, FIG. 4), a labeled downstream probe as defined herein (forexample C, FIG. 4), optionally amplification primers specific for thetarget nucleic acid, a nucleic acid polymerase activity (e.g., a DNApolymerase), a nuclease (e.g. a FEN nuclease) and an appropriate buffer(for example 10× Pfu buffer, Stratagene, Catalog# 200536) in a PCRreaction with the following thermocycling parameters: 95° C. for 2minutes and 40 cycles of 95° C. for 15 sec (denaturation step), 60° C.for 60 sec (annealing step) and 72° C. for 15 sec (extension step).During this reaction an upstream oligonucleotide (for example A, FIG. 4)is extended such that oligonucleotide A partially displaces the 5′labeled end of a downstream oligonucleotide probe according to theinvention (for example oligonucleotide C, FIG. 4) and the resultinglabeled structure is cleaved with a nuclease (e.g., a FEN nuclease)according to the invention. Alternatively, a downstream probe comprisinga secondary structure, as defined herein, (including a stem loop, ahairpin, an internal loop, a bulge loop, a branched structure and apseudoknot) or multiple secondary structures, cloverleaf structures, orany three dimensional structure, as defined herein, can be used.Bi-molecular or multimolecular probes, as defined herein, can also beused. The released labeled fragment is captured by specific binding ofthe binding moiety to a capture element on a solid support according tomethods well known in the art (see Sambrook et al., supra and Ausubel etal., supra). Alternatively, the released labeled fragments or thecleaved downstream probe are directly detected (e.g., cleavage of thedownstream probe between an interactive pair of signal generatingmoieties).

The methods of the invention can also be used in non-PCR basedapplications to detect a target nucleic acid, where such target may beimmobilized on a solid support. Methods of immobilizing a nucleic acidsequence on a solid support are known in the art and are described inAusubel F M et al. Current Protocols in Molecular Biology, John Wileyand Sons, Inc. and in protocols provided by the manufacturers, e.g. formembranes: Pall Corporation, Schleicher & Schuell, for magnetic beads:Dynal, for culture plates: Costar, Nalgenunc, and for other supportsuseful according to the invention, CPG, Inc. A solid support usefulaccording to the invention includes but is not limited to silica basedmatrices, membrane based matrices and beads comprising surfacesincluding, but not limited to any of the solid supports described abovein the section entitled, “Cleavage Structure” and including styrene,latex or silica based materials and other polymers. Magnetic beads arealso useful according to the invention. Solid supports can be obtainedfrom the above manufacturers and other known manufacturers.

The invention also provides for a non-PCR based assay for detecting atarget nucleic acid in solution. The method of the invention can be usedto detect naturally occurring target nucleic acids in solution includingbut not limited to RNA and DNA that is isolated and purified from cells,tissues, single cell organisms, bacteria or viruses. The method of theinvention can also be used to detect synthetic targets in solution,including but not limited to RNA or DNA oligonucleotides, and peptidenucleic acids (PNAs). Non-PCR assays include but are not limited todetection assays involving isothermal linear or exponentialamplification, where the amount of nucleic acid synthesized by the 3′-5′synthetic activity increases linearly or exponentially, and a nuclease(e.g. a FEN nuclease) is used to cleave the displaced strand duringsynthesis. One such example utilizes rolling circle amplification.

In one embodiment of the invention, detection of a nucleic acid targetsequence that is either immobilized or in solution can be performed byincubating an immobilized nucleic acid target sequence or a targetnucleic acid in solution with an upstream oligonucleotide primer that iscomplementary to the target nucleic acid (for example A, FIG. 4) and adownstream oligonucleotide probe having a secondary structure thatchanges upon binding to a target nucleic acid and comprising a bindingmoiety, that is complementary to the target nucleic acid (for example C,FIG. 4), a nuclease (e.g. a FEN nuclease) and a nucleic acid polymerasethat possesses or lacks 5′ to 3′ exonuclease activity. The downstreamprobe is either end labeled at the 5′ or 3′ end, or is labeledinternally. Alternatively, a downstream probe comprising a secondarystructure, as defined herein, (including a stem loop, a hairpin, aninternal loop, a bulge loop, a branched structure and a pseudoknot) ormultiple secondary structures, cloverleaf structures, or any threedimensional structure, as defined herein, can be used. Bi-molecular ormultimolecular probes, as defined herein, can also be used. Detection ofa released labeled fragment that is captured by binding of the bindingmoiety to a capture element involves isotopic, enzymatic, orcolorimetric methods appropriate for the specific label that has beenincorporated into the probe and well known in the art (for example,Sambrook et al., supra, Ausubel et al., supra). Labels useful accordingto the invention and methods for the detection of labels usefulaccording to the invention are described in the section entitled“Cleavage Structure”. Alternatively, the downstream probe furthercomprises a pair of interactive signal generating labeled moieties (forexample a dye and a quencher) that are positioned such that when theprobe is intact, the generation of a detectable signal is quenched, andwherein the pair of interactive signal generating moieties are separatedby a nuclease cleavage site (e.g. a FEN nuclease cleavage site). Inanother embodiment, the downstream probe further comprises a pair ofinteractive signal generating labeled moieties (for example a dye and aquencher) that are positioned such that when the probe is not hybridizedto the target nucleic acid, the generation of a detectable signal isquenched. Upon cleavage by a nuclease (e.g. a FEN nuclease), the twosignal generating moieties are separated from each other and adetectable signal is produced. The presence of a pair of interactivesignal generating labeled moieties, as described above, allows fordiscrimination between annealed, uncleaved probe that may bind to acapture element, and released labeled fragment that is bound to acapture element. Nucleic acid polymerases that are useful for detectingan immobilized nucleic acid target sequence or a nucleic acid targetsequence in solution according to the method of the invention includemesophilic, thermophilic or hyper-thermophilic DNA polymerases lacking5′ to 3′ exonucleolytic activity (described in the section entitled,“Nucleic Acid Polymerases)”. Any nucleic acid polymerase that possess 5′to 3′ exonuclease activity is also useful according to the invention.

According to this non-PCR based method, the amount of a target nucleicacid that can be detected is preferably about 1 pg to μg, morepreferably about 1 pg to 10 ng and most preferably about 1 pg to 10 pg.Alternatively, this non-PCR based method can measure or detectpreferably about 1 molecule to 10²⁰ molecules, more preferably about 100molecules to 10¹⁷ molecules and most preferably about 1000 molecules to10¹⁴ molecules.

The invention also provides for a method of detecting a target nucleicacid in a sample wherein a cleavage structure is formed as described inthe section entitled, “Cleavage Structure”, and the target nucleic acidis amplified by a non-PCR based method including but not limited to anisothermal method, for example rolling circle, Self-sustained SequenceReplication Amplification (3SR), Transcription based amplificationsystem (TAS), and Strand Displacement Amplification (SDA) and anon-isothermal method, for example Ligation chain reaction (LCR). Anuclease (e.g., a FEN nuclease) useful for non-PCR amplification methodswill be active at a temperature range that is appropriate for theparticular amplification method that is employed.

In the amplification protocols described below, samples which need to beprepared in order to quantify the target include: samples, no-templatecontrols, and reactions for preparation of a standard curve (containingdilutions over the range of six orders of magnitude of a solution with adefined quantity of target).

Strand Displacement Amplification (SDA) is based on the ability of arestriction enzyme to nick the unmodified strand of ahemiphosphorothioate form of its recognition site. The appropriate DNApolymerase will initiate replication at this nick and displace thedownstream non-template strand (Walker, 1992, Proc. Natl. Acad. Sci.USA, 89: 392, and PCR Methods and Applications 3: 1-6, 1993). Thepolymerases (Bca and Bst) which are used according to the method of SDAcan also be used in nuclease (e.g. FEN nuclease) directed cleavageaccording to the invention. According to the method of the invention, amolecular beacon is replaced by a nuclease (e.g., a FEN nuclease) activeat 42° C. and a cleavable probe having a secondary structure thatchanges upon binding to a target nucleic acid and further comprising abinding moiety, and comprising a cleavage structure according to theinvention.

A molecular beacon (Mb) is a fluorogenic probe which forms a stem-loopstructure is solution. Typically: 5′-fluorescent dye (e.g. FAM),attached to the 5′-stem region (5-7 nt), the loop region (complementaryto the target, 20 to 30 nt), the 3′-stem region (complementary to the5′-stem region), and the quencher (e.g. DABCYL). If no target ispresent, the MB forms its stem, which brings dye and quencher into closeproximity, and therefore no fluorescence is emitted. When an MB binds toits target, the stem is opened, dye is spatially separated from thequencher, and therefore the probe emits fluorescence (Tyagi S and KramerFR, Nature Biotechnology 14: 303-308 (1996) and U.S. Pat. No.5,925,517).

Strand Displacement Amplification (SDA) is essentially performed asdescribed by Spargo et al., Molecular and Cellular Probes 10: 247-256(1996). The enzymes used include restriction endonuclease BsoBI (NewEngland Biolabs), DNA polymerase 5′-exo-Bca (PanVera Corporation). Thetarget is an insertion-like element (IS6110) found in the Mycobacteriumtuberculosis (Mtb) genome. The primers used are B1: cgatcgagcaagcca (SEQID NO: 35), B2: cgagccgctcgctg (SEQ ID NO: 36), S1:accgcatcgaatgcatgtctcgggtaaggcgtactcgacc (SEQ ID NO: 37) and S2:cgattccgctccagacttctcgggtgtactgagatcccctaccgcatcgaatgcatgtctcgggtaaggcgtactcgacc (SEQ ID NO: 38). TheMycobacterium tuberculosis genomic DNA is serially diluted in humanplacental DNA. SDA is performed in 50 μl samples containing 0 to 1000Mtb genome equivalents, 500 ng human placental DNA, 160 units BsoB1, 8units of 5′exo-Bca, 1.4 mM each dCTPalphaS, TTP, dGTP, DATP, 35 mMK₂PO₄, pH 7.6 0.1 mg/ml acetylated bovine serum albumin (BSA), 3 mMTris-HCl, 10 mM MgCl₂, 11 mM NaCl, 0.3 mM DTT, 4 mM KCl, 4% glycerol,0.008 mM EDTA, 500 nM primers S1 and S2 and 50 nM primers B1 and B2(KCl, glycerol and EDTA are contributed by the BsoB1 storage solution).The samples (35 μl) were heated in a boiling water bath for 3minutes-before the addition of BsoB1 and 5′-exo Bca (10.7 units/lμ BsoB1and 0.53 units/μl 5′-exo Bca in 15 μl of New England Biolabs Buffer 2(20 mM Tris-HCl pH 7.9, 10 mM MgCl₂, 50 mM NaCl, 1 mM DTT). Incubationis at 60° C. for 15 minutes, followed by 5 minutes in a boiling waterbath.

Five μl of each sample in duplicate are removed for detection. Eachreaction contains 1× Cloned Pfu buffer, 3.0 mM MgCl₂, 200 μM of eachdNTP, 5 units exo-Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer: aaggcgtactcgacctgaaa (SEQ ID NO: 39) and fluorogenic probe (forexample FAM-DABCYL): accatacggataggggatctc (SEQ ID NO: 40). Thereactions are subjected to one cycle in a thermal cycler: 2 minutes at95° C., 1 minute at 55° C., 1 minute at 72° C. The fluorescence is thendetermined in a fluorescence plate reader, such as Stratagene'sFluorTracker or PE Biosystems' 7700 Sequence Detection System inPlate-Read Mode. The method of the invention can also be performed witha polymerase that exhibits 5′ to 3′ exonuclease activity and anynuclease included in the section entitled, “Nucleases”.

According to the method of nucleic acid sequence-based amplification(NASBA), molecular beacons are used for quantification of the NASBA RNAamplicon in real-time analysis (Leone, et al., 1998, Nucleic Acids Res.26: 2150). According to the method of the invention, NASBA can becarried out wherein the molecular beacon probe is replaced by a nuclease(e.g. a FEN nuclease) cleavable probe having a secondary structure thatchanges upon binding to a target nucleic acid and comprising a bindingmoiety, and further comprising a cleavage structure according to theinvention and a nuclease (e.g. a FEN nuclease) active at 41° C.

NASBA amplification is performed essentially as described by Leone G, etal., Nucleic Acids Res. 26: 2150-2155 (1998). Genomic, RNA from thepotato leafroll virus (PLRV) is amplified using the PD415 or PD416(antisense) and the PD417 (sense) primers, which are described in detailin Leone G et al., J. Virol. Methods 66: 19-27 (1997). Each NASBAreaction contains a premix of 6 μl of sterile water, 4 μl of 5× NASBAbuffer (5× NASBA buffer is 200 mM Tris-HCl, pH 8.5, 60 mM MgCl₂, 350 mMKCl, 2.5 mM DTT, 5 mM each of dNTP, 10 mM each of ATP, UTP and CTP, 7.5mM GTP and 2.5 mM ITP), 4 μl of 5× primer mix (75% DMSO and 1 μM each ofantisense and sense primers). The premix is divided into 14 μl aliquots,to which 1 μl of PLRV target is added. After incubation for 5 minutes at65° C. and cooling to 41° C. for 5 minutes, 5 μl of enzyme mix is added(per reaction 375 mM sorbitol, 2.1 μg BSA, 0.08 units of RNase H(Pharmacia), 32 units of T7 RNA polymerase (Pharmacia) and 6.4 units ofAMV-RT (Seigakaku)). Amplification is for 90 minutes at 41° C.

In one embodiment, five μl of each sample in duplicate are removed fordetection. Each reaction contains 1× Cloned Pfu buffer, 3.0 mM MgCl₂,200 μM of each dNTP, 5 units exo-Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nMeach upstream primer PD415 or PD416 and the fluorogenic probe (forexample FAM-DABCYL): gcaaagtatcatccctccag (SEQ ID NO: 41). The reactionsare subjected to one cycle in a thermal cycler: 2 minutes at 95° C., 1minute at 55° C., 1 minute at 72° C. The fluorescence in then determinedin a fluorescence plate reader, such as Stratagene's FluorTracker or PEBiosystems' 7700 Sequence Detection System in Plate-Read Mode.

In an alternative embodiment, the detection reaction is performed byadding the fluorogenic probe and Pfu FEN-1 nuclease directly to thenon-PCR based amplification (e.g., NASBA) reaction mixture. Thefluorescent probe is designed to be complementary to a region of thetarget downstream of the promoter region. The RNA polymerase binds tothe promoter region and synthesizes an RNA. The synthesized 3′ end ofthe RNA will form a cleavage structure with the downstream probe whenthe two oligonucleotidesa are sufficiently close. Thus, in this methodamplification and cleavage occur simultaneously. The fluorescence can bedetermined in Stratagene's Mx3005P QPCR System.

In yet another method of the invention, a labeled cleavage structurecomprising an upstream oligonucleotide (e.g., an RNA synthesized by anRNA polymerase) (for example A, FIG. 4), a labeled downstreamoligonucleotide probe and a target nucleic acid (for example B in FIG.4) is formed as described above in the section entitled “CleavageStructure”. In some embodiments, the downstream probe has a secondarystructure that changes upon binding to the target nucleic acid andcomprises a binding moiety (for example C in FIG. 4). Briefly, acleavage structure is formed and cleaved in the presence of a targetnucleic acid, in the presence or absence (depending upon whether the RNApolymerase utilizes a promoter or primer to begin RNA synthesis) of anupstream primer (for example A, FIG. 4), a labeled downstream probe asdefined herein (for example C, FIG. 4) a nucleic acid polymeraseactivity (e.g., a RNA polymerase), a nuclease (e.g. a FEN nuclease) andan appropriate buffer (for example 10×Pfu buffer, Stratagene, Catalog#200536) in a reaction with the following thermocycling parameters: 95°C. for 1-2 minutes followed by cooling to between approximately 40-72°C. During this reaction an upstream oligonucleotide (for example A, FIG.4) is extended by the RNA polymerase such that oligonucleotide Apartially displaces the 5′ labeled end of a downstream oligonucleotideprobe according to the invention (for example oligonucleotide C, FIG. 4)and the resulting labeled structure is cleaved with a nuclease (e.g., aFEN nuclease) according to the invention. Released labeled fragments canbe captured by specific binding of the binding moiety to a captureelement on a solid support according to methods well known in the art(see Sambrook et al., supra and Ausubel et al., supra). Alternatively,the released labeled fragments or the cleaved downstream probe aredirectly detected (e.g., cleavage of the downstream probe between aninteractive pair of signal generating moieties).

Generally, according to these methods wherein amplification occurs by anon-PCR based method, amplification may be carried out in the presenceof a nuclease (e.g. a FEN nuclease), and amplification and cleavage bythe nuclease (e.g. a FEN nuclease) occur simultaneously. Detection ofreleased labeled fragments captured by binding of a binding moiety to acapture element on a solid support is performed as described in thesection entitled “Cleavage Structure” and may occur concurrently with(real time) or after (end-point) the amplification and cleavage processhave been completed.

Endpoint assays can be used to quantify amplified target produced bynon-PCR based methods wherein the amplification step is carried out inthe presence of a nuclease (e.g., a FEN nuclease) (described above).

One may use an in vitro transcription reaction to synthesize RNA from aDNA template present in the reaction. T7-type RNA polymerases, such asT7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase, are commonlyused in such reactions, although many other RNA polymerases may also beused. Usually, but not always, synthesis of RNA is de novo (i.e.,unprimed), and usually, but not always, transcription is initiated at asequence in the template that is specifically recognized by the RNApolymerase, termed a “promoter” or a “promoter region”. A method for invitro transcription is presented herein.

RNA polymerases have been used to amplify target sequences (Krupp, G.,and Soll, D. FEBS Letters (1987) 212:271-275). This approach involvesproduction of a double-stranded copy of the target sequence, insertionof a RNA polymerase promoter sequence, transcription of the copy anddetection by hybridization assay (Kwoh, D. Y., et al., Proc. Natl. Acad.Sci. U.S.A. (1989) 86:1173-1177). Bacteriophage DNA-dependent RNApolymerases (e.g., T3, T7, SP6) have previously been employed for thepreparation in vitro of specific RNA sequences from cloned or syntheticoligonucleotide templates and are well understood (Melton, D. A., etal., Nucleic Acids Res. (1984) 12:7035-7056); Chamberlin, M. and Ryan,T., (1982) in “The Enzymes,” Boyer, P. D., ed., 15:87-108; Martin, C.T., and Coleman, J. E., Biochemistry (1987) 26:2690-2696). Thesepolymerases are highly promoter specific. DNA sequences from numerous T7promoters are known and a consensus sequence has been deduced (Oakley,J. L., and Coleman, J. E., Proc. Natl. Acad. Sci. U.S.A. (1977)74:4266-4270; Dunn, J. J., and Studier, F. W., J. Molec. Biol. (1983)166:477-535). These RNA polymerase based methods can be modified so thata FEN nuclease and detectably labeled probe are added to the reactionmixture so as to allow the amplification and detection reactions toproceed simultaneously. The detectable labeled probe is designed so asto anneal downstream of the targets promoter sequence. Thus, upontranscription the downstream probe and synthesized RNA form a cleavagestructure. The FEN then cleaves the downstream oligonucleotide.

In some embodiments, a promoter is added to the target via apromoter-primer. A number of RNA polymerase promoters may be used forthe promoter region of the promoter-primer. Suitable promoter regionswill be capable of initiating transcription from an operationally linkednucleic acid sequence in the presence of ribonucleotides and an RNApolymerase under suitable conditions. The promoter region will usuallycomprise between about 15 and 250 nucleotides, preferably between about17 and 60 nucleotides, from a naturally occurring RNA polymerasepromoter, a consensus promoter region, or an artificial promoter region,as described in Alberts et al. (1989) in Molecular Biology of the Cell,2d ed. (Garland Publishing, Inc.). Representative promoter regions ofparticular interest include T7, T3 and SP6 as described in Chamberlinand Ryan, The Enzymes (ed. P. Boyer, Academic Press, New York) (1982) pp87-108.

The sequence requirements within the actual promoter for optimaltranscription are generally known in the art as previously described forvarious DNA dependent RNA polymerases, such as in U.S. Pat. Nos.5,766,849 and 5,654,142, and can also be empirically determined.

The promoter-primer oligonucleotides described above may be preparedusing any suitable method known in the art and described herein., suchas, for example, synthesized on an automated DNA synthesizer, e.g. anApplied Biosystems, Inc. Foster City, Calif.)

In yet another aspect, the invention provides a method for detecting atarget nucleic acid utilizing an upstream primer that is extended by anRNA polymerase and a downstream labeled probe. The reaction willgenerally comprise contacting the target nucleic acid with a reactionmixture which includes an RNA polymerase, upstream primer and downstreamprobe and incubating said mixture for an appropriate time and underappropriate conditions to produce a cleavage structure described herein.U.S. patent application Ser. No. 11/217,972, filed Aug. 31, 2005 (hereinincorporated by reference in its entirety) describes RNA amplificationreactions utilizing a primer which is extended by an RNA polymerase.This RNA amplification reaction can be adopted for use in the presentinvention. For example, a downstream labeled probe and FEN nuclease areadded to the reaction described in U.S. patent application Ser. No.11/217,972. In this example, the RNA polymerase will extend thehybridized primer so as to form a cleavage structure with the downstreamprobe.

In this sequence-specific RNA amplification/detection reaction,appropriate primers and probes complementary to the target RNA sequenceare employed along with a FEN nuclease and a suitable RNA polymerasecapable of recognizing the primed RNA molecules, as detailed herein andknown in the art. Amplification, formation of a cleavage structure andcleavage of the cleavage structure is then allowed to proceed undertemperature cycling conditions.

For example, the reaction may be incubate between 70-95° C. to denaturethe target followed by reaction cooling to 35-72° C. During thetemperature reduction, the primers and probe bind to the complementsequences on the target RNA. Once the temperature reaches the optimizedrange, RNA polymerase extends the primer and causes at least the partialdisplacement of the downstream probe, forming a cleavage structure. Thecleavage structure is then cleaved by the nuclease.

Endpoint assays include, but are not limited to the following.

A. Ligation chain reaction (LCR), as described in Landegren, et-al.,1988, Science, 241: 1077 and Barany, PCR Methods and Applications 1:5-169 (1991). An LCR product useful according to the invention will belong enough such that the upstream primer and the labeled downstreamprobe are separated by a gap larger than 8 nucleotides to allow forefficient cleavage by a nuclease (e.g. a FEN nuclease).

B. Self-sustained sequence replication amplification (3SR) Fahy, et al.PCR Methods and Applications 1: 25-33 (1991). Self-Sustained SequenceReplication Amplification (3SR) is a technique which is similar toNASBA. Ehricht R, et al., Nucleic Acids Res. 25: 4697-4699 (1997) haveevolved the 3SR procedure to a cooperatively coupled in vitroamplification system (CATCH). Thus, in one embodiment of the invention,a molecular beacon probe is used for real-time analysis of an RNAamplicon by CATCH. The synthetic target amplified has the sequence:cctctgcagactactattacataatacgactcactatagggatctgcacgtattagcctatagtgagtcgtattaataggaaacaccaaagatgatatttcgtcacagcaagaattcagg (SEQ IDNO: 42). The 3SR reactions contain 40 mM Tris-HCl pH 8.0, 5 mM KCl, 30mM MgCl₂, 1 mM of each dNTP, 1 nM of the double stranded target, 2 μMP1: cctctgcagactactattac (SEQ ID NO: 43) and P2:cctgaattcttgctgtgacg(SEQ ID NO: 44), 5 mM DTT, 2 mM spermidine, 6 units/ul His tagged HIV-1reverse transcriptase, 3 units/ul T7-RNA polymerase and 0.16 units/ulEscherichia coli RNase H. The 100 ul reactions are incubated for 30minutes at 42° C.

Five μl of each sample in duplicate are removed for detection. Eachreaction contains 1× Cloned Pfu buffer, 3.0 mM MgCl₂, 200 μM of eachdNTP, 5 units exo-Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer P1 and fluorogenic probe (for example FAM-DABCYL):taggaaacaccaaagatgatattt (SEQ ID NO: 45). The reactions are subjected toone cycle in a thermal cycler: 2 minutes at 95° C., 1 minute at 55° C.,1 minute at 72° C. The fluorescence in then determined in a fluorescenceplate reader, such as Stratagene's FluorTracker or PE Biosystems' 7700Sequence Detection System in Plate-Read Mode. The method of 3SR can alsobe carried out with a polymerase that exhibits 5′ to 3′ exonucleaseactivity and any nuclease described in the section entitled,“Nucleases”.

C. Rolling circle amplification is described in U.S. Pat. No. 5,854,033and the related Ramification-Extension Amplification Method (RAM) (U.S.Pat. No. 5,942,391). Rolling circle amplification adapted to theinvention is described below.

Real-time assays can also be used to quantify amplified target producedby non-PCR based methods wherein the amplification step is carried outin the presence of a nuclease (e.g. a FEN nuclease) (described above).The method of rolling circle amplification (U.S. Pat. No. 5,854,033) isadapted to include secondary primers for amplification and detection, inconjunction with a nuclease (e.g. a FEN nuclease) and a cleavable probehaving a secondary structure that changes upon binding to a targetnucleic acid and comprising a binding moiety, and further comprising acleavage structure according to the invention, and is carried out attemperatures between 50-600 C. The cleavage pattern of a nuclease (e.g.a FEN nuclease) can be altered by the presence of a single mismatchedbase located anywhere between 1 and 15 nucleotides from the 5′ end ofthe primer wherein the DNA primer is otherwise fully annealed.Typically, on a fully annealed substrate, a nuclease (e.g. a FENnuclease) will exonucleolytically cleave the 5′ most nucleotide.However, a single nucleotide mismatch up to 15 nucleotides in from the5′ end promotes endonucleolytic cleavages. This constitutes a 5′proofreading process in which the mismatch promotes the nuclease actionthat leads to its removal. Thus, the mechanism of nuclease (e.g. FENnuclease) cleavage is shifted from predominantly exonucleolytic cleavageto predominantly endonucleolytic cleavage simply by the presence of asingle mismatched base pair. Presumably this occurs because a mismatchallows a short flap to be created (Rumbaugh et al., 1999, J. Biol.Chem., 274:14602).

The method of the invention can be used to generate a signal indicativeof the presence of a sequence variation in a target nucleic acid,wherein a labeled cleavage structure comprising a fully annealed DNAprimer is formed by incubating a target nucleic acid with a probe havinga secondary structure that changes upon binding to a target nucleic acidand comprising a binding moiety (as described in the section entitled,“Cleavage Structure”) and cleaving the labeled cleavage structure with anuclease (e.g. a FEN nuclease) wherein the release of labeled fragmentscomprising endonucleolytic cleavage products, and the detection ofreleased fragments that are captured by binding of a binding moiety to acapture element on a solid support, is indicative of the presence of asequence variation. Released labeled fragments are detected as describedin the section entitled, “Cleavage Structure”.

V. Samples

The invention provides for a method of detecting or measuring a targetnucleic acid in a sample, as defined herein. As used herein, “sample”refers to any substance containing or presumed to contain a nucleic acidof interest (a target nucleic acid) or which is it self a target nucleicacid, containing or presumed to contain a target nucleic acid ofinterest. The term “sample” thus includes a sample of target nucleicacid (genomic DNA, cDNA or RNA), cell, organism, tissue, fluid orsubstance including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, synovial fluid, urine, tears, stool, externalsecretions of the skin, respiratory, intestinal and genitourinarytracts, saliva, blood cells, tumors, organs, tissue, samples of in vitrocell culture constituents, natural isolates (such as drinking water,seawater, solid materials,) microbial specimens, and objects orspecimens that have been “marked” with nucleic acid tracer molecules.

EXAMPLES

The invention is illustrated by the following nonlimiting exampleswherein the following materials and methods are employed. The entiredisclosure of each of the literature references cited hereinafter areincorporated by reference herein.

Example 1 Probe Design and Preparation

The invention provides for a probe having a secondary structure thatchanges upon binding of the probe to a target nucleic acid andcomprising a binding moiety.

A probe according to one embodiment of the invention is 5-250nucleotides in length and ideally 17-40 nucleotides in length, and has atarget nucleic acid binding sequence that is from 7 to about 140nucleotides, and preferably from 10 to about 140 nucleotides. Probes mayalso comprise non-covalently bound or covalently bound subunits.

One embodiment of a probe comprises a first complementary nucleic acidsequence (for example, b in FIG. 4) and a second complementary nucleicacid sequence (for example, b′ in FIG. 4). In one embodiment wherein theprobe is unimolecular, the first and second complementary nucleic acidsequences are in the same molecule. In one embodiment, the probe islabeled with a fluorophore and a quencher (for example,tetramethylrhodamine and DABCYL, or any of the fluorophore and quenchermolecules described herein (see the section entitled “How To Prepare aLabeled Cleavage Structure”). A probe according to the invention islabeled with an appropriate pair of interactive labels (e.g., a FRETpair or a non-FRET pair). The location of the interactive labels on theprobe is such that an appropriate spacing of the labels on the probe ismaintained to permit the separation of the labels when the probeundergoes a change in the secondary structure of the probe upon bindingto a target nucleic acid. For example, the donor and quencher moietiesare positioned on the probe to quench the generation of a detectablesignal when the probe is not bound to the target nucleic acid.

The probe further comprises a binding moiety (for example ab in FIG. 4,comprising a nucleic acid sequence, i.e., 5′AGCTACTGATGCAGTCACGT3′ (SEQID NO: 26)). In one embodiment of the invention, upon hybridization to atarget nucleic acid, the probe according to the invention, forms acleavage structure comprising a 5′ flap (e.g., ab in FIG. 4). The flapof the cleavage structure thus comprises the binding moiety of theprobe. Cleaving is performed at a cleaving temperature, and thesecondary structure of the probe when not bound to the target nucleicacid is stable at or below the cleaving temperature. Upon cleavage ofthe hybridized probe by a nuclease, the binding moiety is released andbinds specifically to a capture element comprising a nucleic acidsequence; i.e., 5′TCGATGACTACGTCAGTGCA3′ (SEQ ID NO: 27). According tothis embodiment, the binding moiety comprises two regions (for example aand b in FIG. 4). The region of a “binding moiety” that is not a“complementary nucleic acid sequence”, as defined herein, (e.g., a inFIG. 4), is from 1-60 nucleotides, preferably from 1-25 nucleotides andmost preferably from 1-10 nucleotides in length. Region b is one of atleast two complementary nucleic acid sequences of the probe, as definedherein, the length of which is described in detail below.

In one embodiment, in the absence of the target nucleic acid the probefolds back on itself to generate an antiparallel duplex structurewherein the first and second complementary nucleic acid sequences annealby the formation of hydrogen bonds to form a secondary structure. Thesecondary structure of the probe is detected by performing a FRET orfluorescence quenching assay at different temperatures, includingtemperatures that are above and below the Tm of the probe, as describedherein. A probe that exhibits a change in fluorescence that correlateswith a change in temperature (e.g., fluorescence increases as thetemperature of the FRET reaction is increased), greater than a change influorescence simply due to thermal effects on the efficiency offluorophore emission, has secondary structure. Secondary structure iseliminated at a temperature wherein the maximal level of fluorescence isdetected (e.g., fluorescence does not increase above this level atincreased temperatures). The stability of the secondary structure of theprobe is determined in a melting temperature assay or by FRET orfluorescence quenching assay, as described herein.

As a result of the change in the secondary structure of the probe, thebinding moiety becomes accessible for cleavage by a nuclease. In thepresence of the target nucleic acid, and at a temperature that isselected according to the factors that influence the efficiency andselectivity of hybridization of the probe to the target nucleic acid,(e.g., primer length, nucleotide sequence and/or composition, buffercomposition, as described in the section entitled, “Primers and ProbesUseful According to the Invention”) to permit specific binding of theprobe and the target nucleic acid, the probe binds to the target nucleicacid and undergoes a change in the secondary structure. A change in thesecondary structure of the probe can be determined by FRET orfluorescence quenching, as described herein.

In one embodiment, first and second complementary nucleic acid sequencesare 3-25, preferably 4-15 and more preferably 5-11 nucleotides long. Thelength of the first and second complementary nucleic acid sequences isselected such that the secondary structure of the probe when not boundto the target nucleic acid is stable at the temperature at whichcleavage of a cleavage structure comprising the probe bound to a targetnucleic acid is performed. As the target nucleic acid binding sequenceincreases in size up to 100 nucleotides, the length of the complementarynucleic acid sequences may increase up to 15-25 nucleotides. For atarget nucleic acid binding sequence greater than 100 nucleotides, thelength of the complementary nucleic acid sequences are not increasedfurther.

Alternatively, an allele discriminating probe having secondary structureand comprising a binding moiety is prepared.

In one embodiment, an allele discriminating probe according to theinvention preferably comprises a target nucleic acid binding sequencefrom 6 to 50 and preferably from 7 to 25 nucleotides, and sequences ofthe complementary nucleic acid sequences from 3 to 8 nucleotides. Theguanosine-cytidine content of the secondary structure and probe-targethybrids, salt, and assay temperature are considered, for examplemagnesium salts have a strong stabilizing effect, when designing short,allele-discriminating probes.

An allele-discriminating probe with a target nucleic acid bindingsequence near the upper limits of 50 nucleotides long, is designed suchthat the single nucleotide mismatch to be discriminated against occursat or near the middle of the target nucleic acid binding sequence. Forexample, probes comprising a sequence that is 21 nucleotides long arepreferably designed so that the mismatch occurs opposite one of the 14most centrally located nucleotides of the target nucleic acid bindingsequence and most preferably opposite one of the 7 most centrallylocated nucleotides.

Example 2 Probe Design and Preparation

The invention provides for a probe having a secondary structure thatchanges upon binding of the probe to a target nucleic acid andcomprising a binding moiety.

A probe according to one embodiment of the invention is 5-250nucleotides in length and ideally 17-40 nucleotides in length, and has atarget nucleic acid binding sequence that is from 7 to about 140nucleotides, and preferably from 0 to about 140 nucleotides. Probes mayalso comprise non-covalently bound or covalently bound subunits.

One embodiment of a probe comprises a first complementary nucleic acidsequence (for example, b in FIG. 4) and a second complementary nucleicacid sequence (for example, b′ in FIG. 4). In one embodiment wherein theprobe is unimolecular, the first and second complementary nucleic acidsequences are in the same molecule. In one embodiment, the probe islabeled with a fluorophore and a quencher (for example,tetramethylrhodamine and DABCYL, or any of the fluorophore and quenchermolecules described herein (see the section entitled “How To Prepare aLabeled Cleavage Structure”). A probe according to the invention islabeled with an appropriate pair of interactive labels (e.g., a FRETpair or a non-FRET pair). The location of the interactive labels on theprobe is such that an appropriate spacing of the labels on the probe ismaintained to permit the separation of the labels when the probeundergoes a change in the secondary structure of the probe upon bindingto a target nucleic acid. For example, the donor and quencher moietiesare positioned on the probe to quench the generation of a detectablesignal when the probe is not bound to the target nucleic acid.

The probe further comprises a tag comprising the lac repressor protein.In one embodiment of the invention, upon hybridization to a targetnucleic acid, the probe forms a cleavage structure comprising a 5′ flap(e.g., ab in FIG. 4). Cleaving is performed at a cleaving temperature,and the secondary structure of the probe when not bound to the targetnucleic acid is stable at or below the cleaving temperature. Uponcleavage of the hybridized probe by a nuclease, the lac repressorprotein binds specifically to a capture element comprising the doublestranded DNA sequence recognized and bound specifically by the lacrepressor protein:

AATTGTGAGCGGATAACAATT (SEQ ID NO: 4) TTAACACTCGCCTATTGTTAA. (SEQ ID NO:28)

In one embodiment, in the absence of the target nucleic acid the probefolds back on itself to generate an antiparallel duplex structurewherein the first and second complementary nucleic acid sequences annealby the formation of hydrogen bonds to form a secondary structure. Thesecondary structure of the probe is detected by performing a FRET orfluorescence quenching assay at different temperatures, includingtemperatures that are above and below the Tm of the probe, as describedherein. A probe that exhibits a change in fluorescence that correlateswith a change in temperature (e.g., fluorescence increases as thetemperature of the FRET reaction is increased), greater than a change influorescence simply due to thermal effects on the efficiency offluorophore emission, has secondary structure. Secondary structure iseliminated at a temperature wherein the maximal level of fluorescence isdetected (e.g., fluorescence does not increase above this level atincreased temperatures). The stability of the secondary structure of theprobe is determined in a melting temperature assay or by FRET orfluorescence quenching assay, as described herein.

As a result of the change in the secondary structure of the probe, thetag becomes accessible for cleavage by a nuclease. In the presence ofthe target nucleic acid, and at a temperature that is selected accordingto the factors that influence the efficiency and selectivity ofhybridization of the probe to the target nucleic acid, (e.g., primerlength, nucleotide sequence and/or composition, buffer composition, asdescribed in the section entitled, “Primers and Probes Useful Accordingto the Invention”) to permit specific binding of the probe and thetarget nucleic acid, the probe binds to the target nucleic acid andundergoes a change in the secondary structure. A change in the secondarystructure of the probe can be determined by FRET or fluorescencequenching, as described herein.

In one embodiment, first and second complementary nucleic acid sequencesare 3-25, preferably 4-15 and more preferably 5-11 nucleotides long. Thelength of the first and second complementary nucleic acid sequences isselected such that the secondary structure of the probe when not boundto the target nucleic acid is stable at the temperature at whichcleavage of a cleavage structure comprising the probe bound to a targetnucleic acid is performed. As the target nucleic acid binding sequenceincreases in size up to 100 nucleotides, the length of the complementarynucleic acid sequences may increase up to 15-25 nucleotides. For atarget nucleic acid binding sequence greater than 100 nucleotides, thelength of the complementary nucleic acid sequences are not increasedfurther.

Alternatively, an allele discriminating probe having secondary structureand comprising a binding moiety is prepared.

In one embodiment, an allele discriminating probe according to theinvention preferably comprises a target nucleic acid binding sequencefrom 6 to 50 and preferably from 7 to 25 nucleotides, and sequences ofthe complementary nucleic acid sequences from 3 to 8 nucleotides. Theguanosine-cytidine content of the secondary structure and probe-targethybrids, salt, and assay temperature are considered, for examplemagnesium salts have a strong stabilizing effect, when designing short,allele-discriminating probes.

An allele-discriminating probe with a target nucleic acid bindingsequence near the upper limits of 50 nucleotides long, is designed suchthat the single nucleotide mismatch to be discriminated against occursat or near the middle of the target nucleic acid binding sequence. Forexample, probes comprising a sequence that is 21 nucleotides long arepreferably designed so that the mismatch occurs opposite one of the 14most centrally located nucleotides of the target nucleic acid bindingsequence and most preferably opposite one of the 7 most centrallylocated nucleotides.

Example 3

A target nucleic acid can be detected and/or measured by the followingmethod. A labeled cleavage structure is formed prior to the addition ofa FEN nuclease by heating at: 95° C. for 5 minutes and then cooling toapproximately 50-60° C. (a) a sample containing a target nucleic acid (Bin FIG. 4) with (b) an upstream oligonucleotide that specificallyhybridizes to the target nucleic acid, (A, in FIG. 4), and (c) adownstream, 5′ end labeled oligonucleotide probe having a secondarystructure that changes upon binding of the probe to the target nucleicacid and comprising a binding moiety (for example ab in FIG. 4,comprising a nucleic acid sequence, i.e., 5′AGCTACTGATGCAGTCACGT3? (SEQID-NO: 26)), wherein the probe specifically hybridizes to a region ofthe target nucleic acid that is downstream of the hybridizing region ofoligonucleotide A. A polymerase that lacks a 5′ to 3′ exonucleaseactivity but that possesses a 3′ to 5′ DNA synthetic activity, such asthe enzyme a) Yaq exo-, (prepared by mutagenesis using the StratageneQuikChange Site-Directed Mutagenesis kit, catalog number #200518, tomodify Taq polymerase (Tabor and Richardson, 1985, Proc. Natl. Acad.Sci. USA, 82:1074)), a mutant form of Taq polymerase that lacks 5′ to 3′exonuclease activity, b) Pfu, or c) a mutant form of Pfu polymerase thatlacks 3′ to 5′ exonuclease activity (exo-Pfu) is added and incubatedunder conditions that permit the polymerase to extend oligonucleotide Asuch that it partially displaces the 5′ end of oligonucleotide C (forexample 72° C. in 1×Pfu buffer (Stratagene) for 5 minutes to 1 hour. Thedisplaced region of oligonucleotide C forms a 5′ flap that is cleavedupon the addition of a FEN nuclease. Alternatively, extension isperformed with a polymerase that exhibits 5′ to 3′ exonuclease activityand with any nuclease included in the section entitled, “Nucleases”.

A mutant form of Taq polymerase that lacks a 5′ to 3′ exonucleaseactivity but that possesses a 3′ to 5′ DNA synthetic activity comprisesthe following mutation: D144S/F667Y Taq wherein D144S eliminates 5′ to3′ exonuclease activity and F667Y improves ddNTP incorporation.

Exo-mutants of Poll polymerase can be prepared according to the methodof Xu et al., 1997, J. Mol. Biol., 268: 284.

A labeled cleavage structure according to the invention is cleaved witha preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosus FEN-1 that isprepared as described below in Example 9). Cleaving is performed at acleaving temperature, and the secondary structure of the probe when notbound to the target nucleic acid is stable at or below the cleavingtemperature. Cleavage is carried out by adding 2 μl of PfuFEN-1 to a 7μl reaction mixture containing the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x FEN nuclease buffer(10x FEN nuclease buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)2.00 μl PfuFEN-1 enzyme or H₂O 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution (included in the Stratagene Cyclist DNA sequencing kit, catalog#200326), samples are heated at 99° C. for five minutes. Released,labeled, fragments comprising the binding moiety are bound via bindingof the binding moiety to a capture element comprising a nucleic acidsequence, i.e., 5′TCGATGACTACGTCAGTGCA3′ (SEQ ID NO: 27), on a solidsupport. In one embodiment, the labeled fragments are eluted from thecapture element by, for example, decreasing the salt concentration(stringent hybridization conditions typically include saltconcentrations of less than about 1M, more usually less than about 500mM and preferably less than about 200 mM) or by adding an excess ofunlabeled, competitor fragment. Samples containing eluted labeledfragments are analyzed by gel electrophoresis as follows. Samples areloaded on an eleven inch long, hand-poured, 20% acrylamide/bisacrylamide, 7M urea gel. The gel is run at 20 watts until thebromophenol blue has migrated approximately ⅔ the total distance. Thegel is removed from the glass plates and soaked for 10 minutes in fixsolution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (˜80° C.). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

Alternatively, extension is performed with a polymerase that exhibits 5′to 3′ exonuclease activity and with any nuclease included in the sectionentitled, “Nucleases”.

Example 4

A target nucleic acid can be detected and/or measured by the followingmethod. A labeled cleavage structure is formed prior to the addition ofa FEN nuclease by heating at 95° C. for 5 minutes and then cooling toapproximately 50-60° C. (a) a sample containing a target nucleic acid (Bin FIG. 4) with (b) an upstream oligonucleotide that specificallyhybridizes to the target nucleic acid, (A, in FIG. 4), and (c) adownstream, 5′ end labeled oligonucleotide probe having a secondarystructure that changes upon binding of the probe to the target nucleicacid and comprising a lac repressor protein tag, wherein the probespecifically hybridizes to a region of the target nucleic acid that isdownstream of the hybridizing region of oligonucleotide A. A polymerasethat lacks a 5′ to 3′ exonuclease activity but that possesses a 3′ to 5′DNA synthetic activity, such as the enzyme a) Yaq exo-, (prepared bymutagenesis using the Stratagene QuikChange Site-Directed Mutagenesiskit, catalog number #200518, to modify Taq polymerase (Tabor andRichardson, 1985, Proc. Natl. Acad. Sci. USA, 82:1074)), a mutant formof Taq polymerase that lacks 5′ to 3′ exonuclease activity, b) Pfu, orc) a mutant form of Pfu polymerase that lacks 3′ to 5′ exonucleaseactivity (exo-Pfu) is added and incubated under conditions that permitthe polymerase to extend oligonucleotide A such that it partiallydisplaces the 5′ end of oligonucleotide C (for example 720 C in 1×Pfubuffer (Stratagene) for 5 minutes to 1 hour. The displaced region ofoligonucleotide C forms a 5′ flap that is cleaved upon the addition of aFEN nuclease. Alternatively, extension is performed with a polymerasethat exhibits 5′ to 3′ exonuclease activity and with any nucleaseincluded in the section entitled, “Nucleases”.

A mutant form of Taq polymerase that lacks a 5′ to 3′ exonucleaseactivity but that possesses a 3′ to 5′ DNA-synthetic activity comprisesthe following mutation: D144S/F667Y Taq wherein D144S eliminates 5′ to3′ exonuclease activity and F667Y improves ddNTP incorporation.

Exo- mutants of PolI polymerase can be prepared according to the methodof Xu et al., 1997, J. Mol. Biol., 268: 284.

A labeled cleavage structure according to the invention is cleaved witha preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosus FEN-1 that isprepared as described below in Example 9). Cleaving is performed at acleaving temperature, and the secondary structure of the probe when notbound to the target nucleic acid is stable at or below the cleavingtemperature. Cleavage is carried out by adding 2 μl of PfuFEN-1 to a 7μl reaction mixture containing the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x FEN nuclease buffer(10x FEN nuclease buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)2.00 μl PfuFEN-1 enzyme or H₂O 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution (included in the Stratagene Cyclist DNA sequencing kit, catalog#200326), samples are heated at 99° C. for five minutes. Released,labeled, fragments comprising the lac repressor protein are bound viabinding of the lac repressor protein to a capture element comprising thedouble stranded DNA sequence recognized by the lac repressor protein:

(SEQ ID NO: 4) AATTGTGAGCGGATAACAATT (SEQ ID NO: 28)TTAACACTCGCCTATTGTTAA, on a solid support.

In one embodiment, the labeled fragments are eluted from the captureelement by, for example, altering the salt concentration, i.e.,decreasing the salt concentration (stringent hybridization conditionstypically include salt concentrations of less than about 1M, moreusually less than about 500 mM and preferably less than about 200 mM) orby adding an excess of competitor a) lac repressor protein or b) doublestranded DNA sequence recognized by the lac repressor protein. Samplescontaining eluted labeled fragments are analyzed by gel electrophoresisas follows. Samples are loaded on an eleven inch long, hand-poured, 20%acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 watts untilthe bromophenol blue has migrated approximately ⅔ the total distance.The gel is removed from the glass plates and soaked for 10 minutes infix solution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (˜800 C). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

Alternatively, extension is performed with a polymerase that exhibits 5′to 3′ exonuclease activity and with any nuclease included in the sectionentitled, “Nucleases”.

Example 5

A target nucleic acid can be detected and/or measured by the followingmethod. A labeled cleavage structure is formed prior to the addition ofa FEN nuclease by annealing at 95° C. for 5 minutes and then cooling toapproximately 50-60° C. (a) a sample containing a target nucleic acid (Bin FIG. 4) with (b) an upstream oligonucleotide primer that specificallyhybridizes to the target nucleic acid, (A, in FIG. 4), and (c) adownstream, 5′ end labeled oligonucleotide probe having a secondarystructure that changes upon binding of the probe to the target nucleicacid and comprising a binding moiety (for example ab in FIG. 4,comprising a nucleic acid sequence 5′AGCTACTGATGCAGTCACGT3′ (SEQ ID NO:26)), wherein the probe specifically hybridizes to a region of thetarget nucleic acid that is adjacent to the hybridizing region ofoligonucleotide A and further comprises a 5′ region that does nothybridize to the target nucleic acid and forms a 5′ flap. Annealing iscarried out in the presence of 1× Sentinal Molecular beacon core bufferor 10× Pfu buffer.

A labeled cleavage structure according to the invention is cleaved witha preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosus FEN-1 that isprepared as, described below in Example 9). Cleaving is performed at acleaving temperature, and the secondary structure of the probe when notbound to the target nucleic acid is stable at or below the cleavingtemperature. Cleavage is carried out by adding 2 μl of PfuFEN-1 to a 7μl reaction mixture containing the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x FEN nuclease buffer(10x FEN nuclease buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)2.00 μl PfuFEN-1 enzyme or H₂O 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution (included in the Stratagene Cyclist DNA sequencing kit, catalog#200326), samples are heated at 99° C. for five minutes. Released,labeled, fragments comprising a binding moiety are bound via binding ofthe binding moiety to a capture element comprising the sequence,5′TCGATGACTACGTCAGTGCA3′ (SEQ ID NO: 27), on a solid support. In oneembodiment, the labeled fragments are eluted from the capture elementby, for example, decreasing the salt concentration (stringenthybridization conditions typically include salt concentrations of lessthan about 1M, more usually less than about 500 mM and preferably lessthan about 200 mM) or by adding an excess of unlabeled, competitorfragment. Samples containing eluted labeled fragments are analyzed bygel electrophoresis as follows. Samples are loaded on an eleven inchlong, hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. The gelis run at 20 watts until the bromophenol blue has migrated approximately⅔ the total distance. The gel is removed from the glass plates andsoaked for 10 minutes in fix solution (15% methanol, 5% acetic acid) andthen for 10 minutes in water. The gel is placed on Whatmann 3 mm paper,covered with plastic wrap and dried for 2 hours in a heated vacuum geldryer (˜80° C.). The gel is exposed overnight to X-ray film to detectthe presence of a signal that is indicative of the presence of a targetnucleic acid. Alternatively, extension is performed with a polymerasethat exhibits 5′ to 3′ exonuclease activity and with any nucleaseincluded in the section entitled, “Nucleases”.

Example 6

A target nucleic acid can be detected and/or measured by the followingmethod. A labeled cleavage structure is formed prior to the addition ofa FEN nuclease by annealing at 95° C. for 5 minutes and then cooling toapproximately 50-60° C. (a) a sample containing a target nucleic acid (Bin FIG. 4) with (b) an upstream oligonucleotide primer that specificallyhybridizes to the target nucleic acid, (A, in FIG. 4), and (c) adownstream, 5′ end labeled oligonucleotide probe having a secondarystructure that changes upon binding of the probe to the target nucleicacid and comprising a lac repressor protein tag, wherein the probespecifically hybridizes to a region of the target nucleic acid that isadjacent to the hybridizing region of oligonucleotide A and furthercomprises a 5′ region that does not hybridize to the target nucleic acidand forms a 5′ flap. Annealing is carried out in the presence of 1×Sentinal Molecular beacon core buffer or 10× Pfu buffer.

A labeled cleavage structure according to the invention is cleaved witha preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosus FEN-1 that isprepared as described below in Example 9). Cleaving is performed at acleaving temperature, and the secondary structure of the probe when notbound to the target nucleic acid is stable at or below the cleavingtemperature. Cleavage is carried out by adding 2 μl of PfuFEN-1 to a 7μl reaction mixture containing the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x FEN nuclease buffer(10x FEN nuclease buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)2.00 μl PfuFEN-1 enzyme or H₂O 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution (included in the Stratagene Cyclist DNA sequencing kit, catalog#200326), samples are heated at 99° C. for five minutes.

Upon cleavage of the hybridized probe by a nuclease, the lac repressorprotein binds specifically to a capture element comprising the doublestranded DNA sequence recognized by the lac repressor protein:

(SEQ ID NO: 4) AATTGTGAGCGGATAACAATT (SEQ ID NO: 28)TTAACACTCGCCTATTGTTAA, on a solid support.

In one embodiment, the labeled fragments are eluted from the captureelement as described in Example 4, above. Samples containing elutedlabeled fragments are analyzed by gel electrophoresis as follows.Samples are loaded on an eleven inch long, hand-poured, 20%acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 watts untilthe bromophenol blue has migrated approximately ⅔ the total distance.The gel is removed from the glass plates and soaked for 10 minutes infix solution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (˜80° C.). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

Alternatively, extension is performed with a polymerase that exhibits 5′to 3′ exonuclease activity and with any nuclease included in the sectionentitled, “Nucleases”.

Example 7

A target nucleic acid can be detected and/or measured by the followingmethod. A labeled cleavage structure is formed prior to the addition ofa FEN nuclease by annealing at 95° C. for 5 minutes and then cooling toapproximately 50-60° C. (a) a sample containing a target nucleic acid (Bin FIG. 4) with (b) a downstream, 5′ end labeled olignucleotide probehaving a secondary structure that changes upon binding of the probe tothe target nucleic acid and a binding moiety (for example ab in FIG. 4,comprising a nucleic acid sequence, i.e., 5′AGCTACTGATGCAGTCACGT3′ (SEQID NO: 26), wherein the probe specifically hybridizes to a region of thetarget nucleic acid and comprises a 5′ region that does not hybridize tothe target nucleic acid and forms a 5′ flap. Annealing is carried out inthe presence of 1× Sentinal Molecular beacon core buffer or 10× Pfubuffer.

A labeled cleavage structure according to the invention is cleaved witha nuclease that is capable of cleaving this cleavage structure (e.g.,Taq polymerase). Cleaving is performed at a cleaving temperature, andthe secondary structure of the probe when not bound to the targetnucleic acid is stable at or below the cleaving temperature. Cleavage iscarried out by adding 2 μl of a nuclease to a 7 μl reaction mixturecontaining the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x nuclease buffer (500 mMTris-HCl pH 8.0, 100 mM MgCl₂) 2.00 μl nuclease or H₂O 1.3 μl H₂O 7.00μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution (included in the Stratagene Cyclist DNA sequencing kit, catalog#200326), samples are heated at 99° C. for five minutes. Released,labeled, fragments comprising a binding moiety are bound via binding ofthe binding moiety to a capture element comprising a nucleic acidsequence, i.e., 5′TCGATGACTACGTCAGTGCA3′ (SEQ ID NO: 27), on a solidsupport. In one embodiment, the labeled fragments are eluted from thecapture element by, for example, decreasing the salt concentration(stringent hybridization conditions typically include saltconcentrations of less than about 1M, more usually less than about, 500mM and preferably less than about 200 mM) or by adding an excess ofunlabeled, competitor fragment. Samples containing eluted labeledfragments are analyzed by gel electrophoresis as follows. Samples areloaded on an eleven inch long, hand-poured, 20% acrylamide/bisacrylamide, 7M urea gel. The gel is run at 20 watts until thebromophenol blue has migrated approximately ⅔ the total distance. Thegel is removed from the glass plates and soaked for 10 minutes in fixsolution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (˜80° C.). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

Alternatively, extension is performed with a polymerase that exhibits 5′to 3′ exonuclease activity and with any nuclease included in the sectionentitled, “Nucleases”.

Example 8

A target nucleic acid can be detected and/or measured by the followingmethod.

A labeled cleavage structure is formed prior to the addition of a FENnuclease by annealing at 95° C. for 5 minutes and then cooling toapproximately 50-60° C. (a) a sample containing a target nucleic acid (Bin FIG. 4) with (b) a downstream, 5′ end labeled oligonucleotide probehaving a secondary structure that changes upon binding of the probe tothe target nucleic acid and a tag comprising the lac repressor protein,wherein the probe specifically hybridizes to a region of the targetnucleic acid and comprises a 5′ region that does not hybridize to thetarget nucleic acid and forms a 5′ flap. Annealing is carried out in thepresence of 1× Sentinal Molecular beacon core buffer or 10× Pfu buffer.

A labeled cleavage structure according to the invention is cleaved witha nuclease that is capable of cleaving this cleavage structure (e.g.,Taq polymerase). Cleaving is performed at a cleaving temperature, andthe secondary structure of the probe when not bound to the targetnucleic acid is stable at or below the cleaving temperature. Cleavage iscarried out by adding 2 μl of a nuclease to a 7 μl reaction mixturecontaining the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x nuclease buffer (500 mMTris-HCl pH 8.0, 100 mM MgCl₂) 2.00 μl nuclease or H₂O 1.3 μl H₂O 7.00μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution (included in the Stratagene Cyclist DNA sequencing kit, catalog#200326), samples are heated at 99° C. for five minutes. Upon cleavageof the hybridized probe by a nuclease, the lac repressor protein bindsspecifically to a capture element comprising the double stranded DNAsequence recognized by the lac repressor protein:

(SEQ ID NO: 4) AATTGTGAGCGGATAACAATT (SEQ ID NO: 28)TTAACACTCGCCTATTGTTAA, on a solid support.

In one embodiment, the labeled fragments are eluted from the captureelement as described in Example 4, above. Samples containing elutedlabeled fragments are analyzed by gel electrophoresis as follows.Samples are loaded on an eleven inch long, hand-poured, 20%acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 watts untilthe bromophenol blue has migrated approximately ⅔ the total distance.The gel is removed from the glass plates and soaked for 10 minutes infix solution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (a 80° C.). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

Alternatively, extension is performed with a polymerase that exhibits 5′to 3′ exonuclease activity and with any nuclease included in the sectionentitled, “Nucleases”.

Example 9 Cloning Pfu FEN-1

A thermostable FEN nuclease enzyme useful according to the invention canbe prepared according to the following method.

The thermostable FEN nuclease gene can be isolated from genomic DNAderived from P. furiosus (ATCC#43587) according to methods of PCRcloning well known in the art. The cloned PfuFEN-1 can be overexpressedin bacterial cells according to methods well known in the art anddescribed below.

The following pCAL-n-EK cloning oligonucleotides were synthesized andpurified:

a. (SEQ ID NO: 29) 5′GACGACGACAAGATGGGTGTCCCAATTGGTGAGATTATACCAAGAAAA G3′ and b. (SEQ ID NO: 30)5′GGAACAAGACCCGTTTATCTCTTGAACCAACTTTCAAGGGTTGATTGT TTTCCACT 3′.

The Affinity® Protein Expression and Purification System was obtainedfrom Stratagene and used according to the manufacturer's protocols.

Amplification

The insert DNA was prepared by PCR amplification with gene-specificprimers (oligonucleotides a and b, described above) that include 12 and13-nucleotide sequences at the 5′ ends that are complementary to thepCAL-n-EK vector single-stranded tails, thus allowing for directionalcloning. The FEN-1 sequence was amplified from genomic DNA derived fromP. furiosus by preparing amplification reactions (five independent 100μl reactions) containing:

50 μl 10x cPfu Buffer (Stratagene) 7.5 μl Pfu Genomic DNA (approx. 100ng/μl) 7.5 μl PfuTurbo(2.5u/μl), (Stratagene, Catalog # 600250) 15 μlmixed primer pair (100 ng/μl each) (oligonucleotides a and b, describedabove) 4 μl 100 mM dNTP 416 μl H₂O 500 μl totaland carrying out the amplification under the following conditions usinga Stratagene Robocycler 96 hot top thermal cycler:

Window 1 95° C. 1 minute  1 cycle Window 2 95° C. 1 minute 50° C. 1minute 30 cycles 72° C. 3 minutes

The PCR products from each of the five reactions were combined into onetube, purified using StrataPrep PCR and eluted in 50 μl 1 mM Tris-HCl pH8.6. The FEN-1 PCR product was analyzed on a gel and was determined tobe approximately 1000 bp.

The PCR product comprising the fen-1 gene was cloned into the pCALnEKLIC vector (Stratagene) by creating ligation independent cloning termini(LIC), annealing the PCR product comprising the fen-1 gene to thepCALnEK LIC vector (Stratagene), and transforming cells with theannealing mixture according to the following method. Briefly, followingPCR amplification, the PCR product is purified and treated with Pfu DNApolymerase in the presence of dATP (according to the manual includedwith the Affinity® Protein Expression and Purification System,Stratagene, catalog #200326). In the absence of dTTP, dGTP and dCTP, the3′ to 5′-exonuclease activity of Pfu DNA polymerase removes at least 12and 13 nucleotides at the respective 3′ ends of the PCR product. Thisactivity continues until the first adenine is encountered, producing aDNA fragment with 5′-extended single-stranded tails that arecomplementary to the single-stranded tails of the pCAL-n-EK vector.

Creating LIC termini.

LIC termini were created by preparing the following mixture:

45 μl purified PCR product (~0.5 μg/μl) 2.5 μl 10 mM dATP. 5 μl 10x cPfubuffer 1 μl cPfu (2.5u/μl) 0.5 μl H₂O

cPfu and cPfu buffer can be obtained from Stratagene (cPfu, StratageneCatalog #600153 and cPfu buffer, Stratagene Catalog #200532).

Samples were incubated at 72° C. for 20 minutes and products were cooledto room temperature. To each sample was added 40 ng prepared pCALnEK LICvector (the prepared vector is available commercially from Stratagene inthe Affinity LIC Cloning and Protein Purification Kit (214405)). Thevector and insert DNA are combined, allowed to anneal at roomtemperature and transformed into highly competent bacterial host cells(Wyborski et al., 1997, Strategies, 10:1).

Preparing Cells for Production of FEN

Two liters of LB-AMP was inoculated with 20 ml of an overnight cultureof a FEN-1 clone (clone 3). Growth was allowed to proceed forapproximately 11 hours at which point cells had reached an OD₆₀₀=0.974.Cells were induced overnight (about 12 hours) with 1 mM IPTG. Cells werecollected by centrifugation and the resulting cell paste was stored at−20° C.

Purification of Tagged FEN-1

Cells were resuspended in 20 ml of Calcium binding buffer

CaCl₂ binding Buffer 50 mM Tris-HCl (pH 8.0) 150 mM NaCl 1.0 mM MgOAc 2mM CaCl₂

The samples were sonicated with a Branson Sonicator using a microtip.The output setting was 5 and the duty cycle was 90%. Samples weresonicated three times and allowed to rest on ice during the intervals.The sonicate was centrifuged at 26,890×g. Cleared supernatants weremixed with 1 ml of washed (in CaCl₂ binding buffer) calmodulin agarose(CAM agarose) in a 50 ml conical tube and incubated on a slowly rotatingwheel in a cold room (4° C.) for 5 hours. The CAM agarose was collectedby light centrifugation (5000 rpm in a table top centrifuge).

Following removal of the supernatant, the CAM agarose was washed with 50ml CaCl₂ binding buffer and transferred to a disposable drip column. Theoriginal container and pipet were rinsed thoroughly to remove residualagarose. The column was rinsed with approximately 200 ml of CaCl₂binding buffer.

Elution was carried out with 10 ml of 50 mM NaCl elution buffer (50 mMNaCl, 50 mM Tris-HCl pH 8.0, 2 mM EGTA). 0.5 ml fractions werecollected. A second elution step was carried out with 1M NaCl elutionbuffer wherein 0.5 ml fractions were collected.

Evaluation of Purified Tagged FEN-1

Fractions containing CBP-tagged Pfu FEN-1 eluted in 1M NaCl were boiledin SDS and analyzed by SDS-PAGE on a 4-20% gel stained with Sypro Orange(FIG. 5).

The protein concentration of uncleaved FEN-1 was determined to beapproximately 150 ng/microliter (below).

Enterokinase Protease (EK) Cleavage of the Purified FEN-1

Fractions 3-9 were dialyzed in 50 mM NaCl, 50 mM Tris-HCl pH 8.0 and 2mM CaCl₂ overnight at 4° C.

An opaque, very fine precipitate appeared in the dialyzed FEN-1. Whenthe sample was diluted 1/20 the precipitate was removed. When the samplewas diluted 1/3 insoluble material was still detectable. The 1/3 dilutedmaterial was heated at 37° C. for 2 minutes and mixed with Tween 20 to afinal concentration of 0.1%. Upon the addition of the Tween 20, therewas an almost immediate formation of “strings” and much coarser solidsin the solution which could not be reversed even after the solution wasadjusted to 1 M NaCl.

EK cleavage was carried out using as a substrate the sample that wasdiluted 1/20 as well as with a dilute sample prepared by rinsing thedialysis bag with 1× EK buffer.

EK cleavage was carried out by the addition of 1 μl EK (1 u/μl)overnight at room temperature (about 16 hours).

100 μl of STI agarose combined with 100 μl of CAM agarose were rinsedtwice with 10 ml of 1×STI buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 2mM CaCl₂, 0.1% Tween 20). NaCl was added to the two EK samples to bringthe final concentration to 200 mM NaCl. The two samples were combinedand added to the rinsed agarose. The samples were rotated slowly on awheel at 4° C. for three hours and separated by light centrifugation ina table top centrifuge (as described). The supernatant was removed andthe resin was rinsed twice with 500 μl 1× STI. The two rinses werecombined and saved separately from the original supernatant. Sampleswere analyzed by SDS-PAGE on a 4-20% gel.

The concentration of digested product was approximately 23 ng/μl asdetermined by comparison to a Pfu standard at a concentration ofapproximately 50 ng/ml.

Example 10 FEN Nuclease Activity

The endonuclease activity of a FEN nuclease and the cleavage structurerequirements of a FEN nuclease prepared as described in Example 2 can bedetermined according to the methods described either in the sectionentitled “FEN nucleases” or below.

Briefly, three templates (FIG. 2) are used to evaluate the activity of aFEN nuclease according to the invention. Template 1 is a 5′ ³³P labeledoligonucleotide (Heltest4) with the following sequence:

(SEQ ID NO: 1) 5′AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG3′.

The underlined section of Heltest4 represents the region complementaryto M13 mp18+. The cleavage product is an 18 nucleotide fragment with thesequence AAAATAAATAAAAAAAAT (SEQ ID NO: 2). Heltest4 binds to M13 toproduce a complementary double stranded domain as well as anon-complementary 5′ overhang. This duplex forms template 2 (FIG. 2).Template 3 (FIG. 2) has an additional primer (FENAS) bound to M13 whichis directly adjacent to Heltest 4. The sequence of FENAS is:5′CCATTCGCCATTCAGGCTGCGCA 3′ SEQ ID NO: 3). In the presence of template3, a FEN nuclease binds the free 5′ terminus of Heltest4, migrates tothe junction and cleaves Heltest4 to produce an 18 nucleotide fragment.The resulting cleavage products are separated on a 6% acrylamide, 7Murea sequencing gel.

Templates are prepared as described below:

Template 1 Template 2 Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl14 μl FENAS ** ** 14 μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl  4.6 μl 4.6 μl 

Pfu buffer can be obtained from Stratagene (Catalog #200536).

The template mixture is heated at 95° C. for five minutes, cooled toroom temperature for 45 minutes and stored at 4° C. overnight.

The enzyme samples are as follows:A. H₂O (control)B. 2 μl undiluted uncleaved FEN-1 (˜445 ng/μl)C. 2 μl 1/10 dilution of uncleaved FEN-1 (˜44.5 ng/μl)D. 2 μl enterokinase protease (EK) cleaved FEN-1 (˜23 ng/μl)

The four reaction mixtures are mixed with the three templates asfollows:

3 μl template 1, template 2 or template 3 0.7 μl 10x cloned Pfu buffer0.6 μl 100 mM MgCl₂ 2.00 μl FEN-1 or H₂O 0.7 μl H₂O 7.00 μl total volume

The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide 7M urea CastAway gel (Stratagene).

Alternatively, FEN nuclease activity can be analyzed in the followingbuffer wherein a one hour incubation time is utilized.

10x FEN Nuclease Buffer 500 mM Tris-HCl pH 8.0 100 mM MgCl₂

The reaction mixture is as follows:

3 μl template 1, template 2 or template 3 0.7 μl 10x FEN nuclease buffer2.00 μl FEN-1 or H₂O (A-D, above) 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 50° C. in the Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop (95%formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol,available from Stratagene) dye solution, samples are heated at 99° C.for five minutes. Samples are loaded on an eleven inch long,hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. The gel is runat 20 watts until the bromophenol blue has migrated approximately ⅔ thetotal distance. The gel is removed from the glass plates and soaked for10 minutes in fix solution (15% methanol, 5% acetic acid) and then for10 minutes in water. The gel is placed on Whatmann 3 mm paper, coveredwith plastic wrap and dried for 2 hours in a heated vacuum gel dryer(˜80° C.). The gel is exposed overnight to X-ray film.

An autoradiograph of a FEN-1 nuclease assay wherein templates 1, 2 and 3(prepared as described above) are cleaved by the addition of:

A. H₂O B. 2 μl of CBP-tagged Pfu FEN-1

C. 2 μl of CBP-tagged Pfu FEN-1 diluted (1:10)D. 2 μl of EK cleaved Pfu FEN-1 is presented in FIG. 6.

The lanes are as follows. Lanes 1A, 1B, 1C and 1D represent template Icleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1: 10 dilution ofCBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively. Lanes 2A,2B, 2C and 2D represent template 2 cleaved with H₂O, undilutedCBP-tagged Pfu FEN-1, a 1:10 dilution of CBP-tagged Pfu FEN-1 and EKcleaved Pfu FEN-1, respectively. Lanes 3A, 3B, 3C and 3D representtemplate 3 cleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1:10dilution of CBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively.

Tagged Pfu FEN-1 contains the N-terminal CBP affinity purification tag.Any differences in activity between tagged and untagged versions ofFEN-1 are due to differences in protein concentration (concentrations ofenzyme samples are provided above) since the amounts of tagged versusuntagged FEN-1 are not equivalent. Both tagged and untagged Pfu FEN-1demonstrate cleavage activity.

FIG. 6 demonstrates the background level of cleavage in the absence ofFEN-1 (lanes 1A, 2A and 3A). Further, this figure demonstrates thattagged Pfu FEN-1 cleaves more of template 2 as compared to template 1.In particular, the greatest amount of template 2 is cleaved in thepresence of undiluted, tagged Pfu FEN-1 (lane 2B). Analysis of template3 demonstrates that the greatest amount of template 3 is cleaved byundiluted, tagged Pfu FEN-1 and the least amount of template 3 iscleaved by diluted tagged FEN-1. Labeled probe migrates as a 40-43nucleotide band. FEN-1 preferentially cleaves template 3 (whichcomprises an upstream primer) as compared to template 2. The cleavageproduct bands are the major bands migrating at 16-20 nucleotides.Heterogeneity in the labeled cleavage products is the result ofheterogeneity in the labeled substrate, which was not gel-purified priorto use.

Example 11 PCR Amplification and Detection of β-Actin in the Presence ofa FEN-1 Nuclease and a Tag Polymerase Deficient in 5′ to 3′ ExonucleaseActivity

A PCR assay is used to detect a target nucleic acid. According to themethod of this assay, a PCR reaction is carried out in the presence of aprobe having a secondary structure that changes upon binding to a targetnucleic acid and comprising a binding moiety or a tag, Taq polymerasedeficient in 5′ to 3′ exonuclease activity (for example Yaq exo-), and athermostable FEN-1 nuclease (e.g. Pfu FEN-1, prepared as described inExample 2). Detection of the release of fluorescently labeled fragmentsthat bind, via binding of the binding moiety or tag, to a captureelement on a solid support indicates the presence of the target nucleicacid.

Duplicate PCR reactions containing 1× Sentinel Molecular beacon corebuffer, 3.5 mM MgCl₂, 200 μM of each dNTP, a Taq polymerase deficient in5′ to 3′ exonuclease activity (˜1.45 U), Pfu FEN-1 (˜23 ng), β-Actinprimers (300 nM each) and β-actin specific fluorogenic probe having asecondary structure that changes upon binding of the probe to theβ-Actin target sequence and comprising a binding moiety or tag. 10 ng ofhuman genomic DNA (Promega) is used as the target nucleic acid in eachreaction. This reaction is performed in a 50 μl volume. Negative controlreactions containing either Pfu FEN-1 alone, a Taq polymerase deficientin 5′ to 3′ exonuclease activity alone or reaction mixtures containingall components except a human genomic DNA template are prepared.Positive control reactions comprising 2.5 Units of Taq 2000 are alsoprepared. During the PCR reaction, there is simultaneous formation of acleavage structure, amplification of the β-actin target sequence andcleavage of the cleavage structure. Thermocycling parameters areselected such that cleavage of the cleavage structure is performed at acleaving temperature, and the secondary structure of the probe, when notbound to the target nucleic acid is stable at or below the cleavingtemperature. Reactions are assayed in a spectrofluorometric thermocycler(ABI 7700). Thermocycling parameters are 95° C. for 2 min and 40 cyclesof 95° C. for 15 sec, 60° C. for 60 sec and 72° C. for 15 sec. Samplesare interrogated during the annealing step.

Released, fluorescently labeled fragments are bound, via the bindingmoiety or tag present on the probe, to a capture element bound to asolid support.

Example 12 PCR Amplification and Detection of β-Actin in the Presence ofa FEN-1 Nuclease and a Pfu Polymerase Deficient in 5′ to 3′ ExonucleaseActivity

A PCR assay is used to detect a target nucleic acid. According to themethod of this assay, a PCR reaction is carried out in the presence of aprobe having a secondary structure that changes upon binding of theprobe to the β-actin target nucleic acid and comprising a binding moietyor tag, Pfu polymerase (naturally lacking 5′ to 3′ exonuclease activity)or, in addition, Pfu polymerase deficient in 3′ to 5′ exonucleaseactivity as well (for example exo-Pfu), and a thermostable FEN-1nuclease (Pfu FEN-1). Detection of the release of fluorescently labeledfragments that bind, via binding of the binding moiety or tag, to acapture element on a solid support indicates the presence of the targetnucleic acid.

Duplicate PCR reactions containing 1× Cloned Pfu buffer (available fromStratagene, Catalog #200532), 3.0 mM MgCl₂, 200 μM of each dNTP, 5 unitsof a Pfu polymerase deficient in 3′ to 5′ exonuclease activity, taggedor untagged Pfu FEN-1 (˜23 ng), PEF (1 ng) (described in WO 98/42860),β-Actin primers. (300 nM each), and fluorogenic probe having a secondarystructure that changes upon binding of the probe to the target β-actinnucleic acid sequence are prepared. 10 ng of human genomic DNA (Promega)is used as the target nucleic acid in each reaction. Reactions areperformed in a 50 μl volume. Negative control reactions comprising a Pfupolymerase deficient in both 5′ to 3′ and 3′ to 5′ exonucleaseactivities alone or containing all of the components except the humangenomic DNA template are also prepared. A reaction mixture containing2.5 Units of Taq 2000 is prepared and used as a positive control. Duringthe PCR reaction, there is simultaneous formation of a cleavagestructure, amplification of the β-actin target sequence and cleavage ofthe cleavage structure. Thermocycling parameters are selected such thatcleavage of the cleavage structure is performed at a cleavingtemperature, and the secondary structure of the probe, when not bound tothe target nucleic acid is stable at or below the cleaving temperature.Reactions are analyzed in a spectrofluorometric thermocycler (ABI 7700).Thermocycling parameters are 95° C. for 2 min and 40 cycles of 95° C.for 15 sec, 60° C. for 60 sec and 72° C. for 15 sec.

Released, fluorescently labeled fragments are bound via the bindingmoiety or tag present on the probe, to a capture element bound to asolid support.

Example 13

An assay according to the invention involving rolling circleamplification is performed using the human ornithine transcarbamylasegene as a target, which is detected in human DNA extracted from buffycoat by standard procedures. Target (400 ng) is heat-denatured for 4minutes at 97° C., and incubated under ligation conditions in thepresence of two 5′-phosphorylated oligonucleotides, an open circle probeand one gap oligonucleotide. The open circle probe has the sequence:gaggagaataaaagtttctcataagactcgtcatgtctcagcagcttctaacggtcactaatacgactcactataggttctgcctctgggaacac (SEQ ID NO: 46), the gap nucleotide for the wild-type sequenceis: tagtgatc. FIGS. 7 and 8 depict rolling circle probes and rollingcircle amplification. The reaction buffer (40 ul) contains 5 units/μl ofT4 DNA ligase (New England Biolabs), 10 mM Tris-HCl, pH 7.5, 0.2 M NaCl,10 mM MgCl₂, 4 mM ATP, 80 nM open circle probe and 100 nM gapoligonucleotide. After incubation for 25 minutes at 37° C., 25 ul areremoved and added to 25 ul of a solution containing 50 mM Tris-HCl, pH7.5, 10 mM MgCl₂, 1 mM DTT, 400 μM each of dTTP, dATP, dGTP, dCTP, 0.2μM rolling circle replication primer: gctgagacatgacgagtc (SEQ ID NO:47), phi29 DNA polymerase (160 ng/50 ul). The sample is incubated for 30minutes at 30° C.

RNA is produced from a T7 promoter present in the open circle probe, bythe addition of a compensating buffer (a stock solution or concentrate)that is diluted to achieve the following concentration of reagents: 35mM Tris-HCl, pH 8.2, 2 mM spermidine, 18 mm MgCl₂, 5 mM GMP, 1 mM ofATP, CTP, GTP, 333 uM UTP, 667 uM Biotin-16-UTP, 0.03% Tween 20, 2 unitsper ul of T7 RNA polymerase. RNA production is performed as described inU.S. Pat. No. 5,858,033. The incubation is allowed to proceed for 90minutes at 37° C.

Five μl of each sample (the actual test sample, a (−) ligase controlsample, a (−) phi29 DNA polymerase control and a (−)T7 RNA polymerasecontrol) in duplicate are removed for detection. The reversetranscription process includes the steps of A) ligating the open circle,B) synthesizing rolling circle single stranded DNA, C) making RNA (froma T7 promoter present in the open circle probe), D) reverse transcribingthe RNA to make cDNA, and E) performing PCR amplification of the cDNAusing primers and probes for generation of an detection of FEN cleavagestructures, according to the invention. For reverse transcription, thereagents and protocols supplied with the Stratagene Sentinel Single-TubeRT-PCR Core Reagent Kit (Cat# 600505) are used, except for thesubstitution of equal amounts of Yaq DNA polymerase for the Taq 2000 DNApolymerase which is recommended by the manufacturer. Each reactioncontains 1× Sentinel molecular beacon RT-7PCR core buffer, 3.5 mM MgCl₂,200 μM of each dNTP, 5 units exo-Pfu 23 ng Pfu FEN-1, 1 ng PEF, 500 nMeach of the upstream primer: aagtttctcataagactcgtcat (SEQ ID NO: 48),the reverse primer: aggcagaacctatagtgagtcgt (SEQ ID NO: 49), and thefluorogenic probe (for example labeled with FAM-DABCYL) having asecondary structure, as defined herein, that changes upon binding to thetarget nucleic acid and further comprising a binding moiety. Thereactions are subjected to incubation for 30 minutes at 45° C., 3minutes at 95° C., followed by one cycle in a thermal cycler: 2 minutesat 95° C., 1 minute at 50° C., 1 minute at 72° C. The fluorescence inthen determined in a fluorescence plate reader, such as Stratagene'sFluorTracker or PE Biosystems' 7700 Sequence Detection System inPlate-Read Mode.

A crosscheck for the efficiency of detection is possible because of theincorporation of Biotin-16-UTP in the rolling circle amplification RNAproduct. An aliquot of the reactions is captured on glass slides (oralternatively in microwell plates) using an immobilized capture probe.Detection of the captured RNA amplicon is described in detail in U.S.Pat. No. 5,854,033, hereby incorporated by reference.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing detailed description is providedfor clarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

1. A composition for generating a signal indicative of the presence of atarget nucleic acid sequence in a sample, said composition comprising anRNA polymerase, a FEN nuclease, a primer and a probe.
 2. The compositionof claim 1, wherein the FEN nuclease is a flap-specific nuclease.
 3. Thecomposition of claim 1, wherein the FEN nuclease is thermostable.
 4. Thecomposition of claim 1, wherein the probe comprises at least one labeledmoiety capable of providing a signal.
 5. The composition of claim 1,wherein the RNA polymerase is T7-RNA polymerase, SP6-RNA polymerase, T3RNA polymerase and NS5B RNA polymerase.
 6. A composition for performinga non-polymerase chain reaction process for simultaneously forming acleavage structure comprising duplex and single-stranded nucleic acid,amplifying a target nucleic acid sequence in a sample and cleaving saidcleavage structure, the composition comprising an RNA polymerase, a FENnuclease, a labeled probe.
 7. The composition of claim 6, wherein theFEN nuclease is a flap-specific nuclease.
 8. The composition of claim 6,wherein the FEN nuclease is thermostable.
 9. The composition of claim 6,wherein the probe comprises at least one labeled moiety capable ofproviding a signal.
 10. The composition of claim 6, wherein the RNApolymerase is T7-RNA polymerase, SP6-RNA polymerase, T3 RNA polymeraseor NS5B RNA polymerase
 11. A kit for generating a signal indicative ofthe presence of a target nucleic acid sequence in a sample, the kitcomprising a FEN nuclease, an RNA polymerase, a probe and a suitablebuffer.
 12. The kit of claim 11, wherein the FEN nuclease is aflap-specific nuclease.
 13. The kit of claim 11, wherein the FENnuclease is thermostable.
 14. The kit of claim 11, wherein the probecomprises at least one labeled moiety capable of providing a signal. 15.The kit of claim 11, wherein the RNA polymerase is T7-RNA polymerase,SP6-RNA polymerase, T3 RNA polymerase or NS5B RNA polymerase
 16. The kitof claim 11, wherein said FEN nuclease, said buffer and said RNApolymerase are in the same composition.
 17. A composition for generatinga signal indicative of the presence of a target nucleic acid sequence ina sample, the composition comprising an RNA polymerase, a FEN nuclease,and a probe that is complementary to the target nucleic acid.
 18. Thecomposition of claim 17, further comprising the target nucleic acid. 19.The composition of claim 17, wherein the probe has a 5′ flap.
 20. Thecomposition of claim 19, wherein the 5′ region of the probe is noncomplementary to the target nucleic acid.
 21. The composition of claim17; wherein the probe comprises: at least one labeled moiety capable ofproviding a signal.
 22. The composition of claim 17, wherein the probecomprises a pair of interactive signal generating labeled moietieseffectively positioned to quench the generation of a detectable signal,the labeled moieties being separated by a site susceptible to FENnuclease cleavage.
 23. The composition of claim 22, wherein the pair ofinteractive signal generating moieties comprises a quencher moiety and afluorescent moiety.
 24. The composition of claim 17, further comprisingan upstream oligonucleotide primer, wherein the upstream oligonucleotideprimer hybridizes upstream of the probe and is extended by the RNApolymerase.
 25. The composition of claim 24, further comprising areverse primer.
 26. The composition of claim 17, wherein the FENnuclease is selected from the group consisting of FEN nuclease enzymederived from Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcusfuriosus, Taq, Tfl and Bca.
 27. The composition of claim 17, wherein theRNA polymerase is an RNA dependent polymerase.
 28. The composition ofclaim 17, wherein the RNA polymerase is a DNA dependent polymerase. 29.The composition of claim 17, wherein the RNA polymerase is selected fromthe group consisting of: T7-RNA polymerase, SP6-RNA polymerase, T3 RNApolymerase, and NS5B RNA polymerase.
 30. The composition of claim 17,wherein the RNA polymerase is thermostable.
 31. A kit for generating asignal indicative of the presence of a target nucleic acid sequence in asample, the composition comprising an RNA polymerase, a FEN nuclease,and a probe that is complementary to the target nucleic acid.
 32. Thekit of claim 31, wherein the probe comprises at least one labeled moietycapable of providing a signal.
 33. The kit of claim 31, wherein theprobe comprises a pair of interactive signal generating labeled moietieseffectively positioned to quench the generation of a detectable signal,the labeled moieties being separated by a site susceptible to FENnuclease cleavage.
 34. The kit of claim 33, wherein the pair ofinteractive signal generating moieties comprises a quencher moiety and afluorescent moiety.
 35. The kit of claim 31, further comprising anupstream oligonucleotide primer, wherein the upstream oligonucleotideprimer hybridizes upstream of the probe and is extended by said RNApolymerase.
 36. The kit of claim 35, further comprising a reverseprimer.
 37. The kit of claim 31, wherein the FEN nuclease is selectedfrom the group consisting of FEN nuclease enzyme derived fromArchaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus,Taq, Tfl and Bca.
 38. The kit of claim 31, wherein the RNA polymerase isT7-RNA polymerase, SP6-RNA polymerase, T3 RNA polymerase and NS5B RNApolymerase.
 39. The kit of claim 31, wherein the RNA polymerase isthermostable.
 40. The kit of claim 31, wherein the RNA polymerase is anRNA dependent RNA polymerase.
 41. The kit of claim 31, wherein the RNApolymerase is a DNA dependent RNA polymerase.
 42. A method of generatinga signal indicative of the presence of a target nucleic acid sequence ina sample, comprising forming a cleavage structure by incubating a samplecomprising a target nucleic acid sequence with an RNA polymerase andcleaving the cleavage structure with a FEN nuclease to generate asignal, wherein generation of the signal is indicative of the presenceof a target nucleic acid sequence in the sample.
 43. A method ofdetecting or measuring a target nucleic acid sequence, comprisingforming a cleavage structure by incubating a sample comprising a targetnucleic acid sequence with an RNA polymerase, cleaving the cleavagestructure with a FEN nuclease to release a nucleic acid fragment anddetecting and/or measuring the release of the fragment as an indicationof the presence of the target sequence in the sample.
 44. The method ofclaim 42, wherein the RNA polymerase is thermostable
 45. The method ofclaim 42, wherein the RNA polymerase is T7-RNA polymerase, SP6-RNApolymerase, T3 RNA polymerase or NS5B RNA polymerase.
 46. The method ofclaim 42, wherein the FEN nuclease is a flap-specific nuclease.
 47. Themethod of claim 42, wherein the FEN nuclease is thermostable.
 48. Themethod of claim 42, wherein a cleavage structure is formed comprising atleast one labeled moiety capable of providing a signal.
 49. The methodof claim 42, wherein a cleavage structure is formed comprising a pair ofinteractive signal generating labeled moieties effectively positioned toquench the generation of a detectable signal, the labeled moieties beingseparated by a site susceptible to FEN nuclease cleavage, therebyallowing the nuclease activity of the FEN nuclease to separate the firstinteractive signal generating labeled moiety from the second interactivesignal generating labeled moiety by cleaving at the site susceptible toFEN nuclease, thereby generating a detectable signal.
 50. The method ofclaim 49, wherein the pair of interactive signal generating moietiescomprises a quencher moiety and a fluorescent moiety.
 51. The method ofclaim 42, wherein a cleavage structure comprises an RNA extensionproduct and a downstream probe with a 5′ flap.
 52. A method fordetecting a target nucleic acid sequence in a sample, comprising: mixinga probe, a target nucleic acid having a promoter region, a FEN nucleaseand an RNA polymerase, under conditions which are permissive for thesteps of (i) binding of the RNA polymerase to the promoter region in thetarget nucleic acid sequence and annealing of the probe to the target,(ii) synthesizing an RNA polymerase extension product, (iii) forming acleavage structure, and (iv) and cleaving the cleavage structure withthe FEN nuclease and detecting and/or measuring the release of labeledfragments or cleavage of the probe as an indication of the presence ofthe target sequence in the sample.
 53. A method for detecting a targetnucleic acid sequence in a sample, comprising: mixing a probe, a targetnucleic acid, a FEN nuclease, a primer and an RNA polymerase, underconditions which are permissive for the steps of (i) annealing of theprimer and probe to the target nucleic acid, (ii) synthesizing an RNApolymerase extension product from said primer, (iii) forming a cleavagestructure, and (iv) and cleaving the cleavage structure with the FENnuclease and detecting and/or measuring the release of labeled fragmentsor cleavage of the probe as an indication of the presence of the targetsequence in the sample.
 54. The method of claim 52 or 53, wherein theFEN nuclease is selected from the group consisting of FEN nucleaseenzyme derived from Archaeglobus fulgidus, Methanococcus jannaschii,Pyrococcus furiosus, Taq, Tfl and Bca.
 55. The method of claim 52 or 53,wherein the RNA polymerase is thermostable.
 56. The method of claim 52or 53, wherein the RNA polymerase is T7-RNA polymerase, SP6-RNApolymerase, T3 RNA polymerase and NS5B RNA polymerase.
 57. The method ofclaim 52 or 53, wherein the probe comprises at least one labeled moietycapable of providing a signal.
 58. The method of claim 52 or 53, whereinthe probe comprises a pair of interactive signal generating labeledmoieties effectively positioned to quench the generation of a detectablesignal, the labeled moieties being separated by a site susceptible toFEN nuclease cleavage, thereby allowing the nuclease activity of the FENnuclease to separate the first interactive signal generating labeledmoiety from the second interactive signal generating labeled moiety bycleaving at the site susceptible to FEN nuclease, thereby generating adetectable signal.
 59. The method of claim 58, wherein the pair ofinteractive signal generating moieties comprises a quencher moiety and afluorescer moiety.
 60. The method of claim 52 or 53, wherein the step ofdetecting and/or measuring the release of labeled fragments comprisesdetecting a change in fluorescence between an interactive pair oflabels.
 61. The method of claim 52 or 53, wherein the RNA polymerasesdisplaces at least a portion of the downstream probe.
 62. The method ofclaim 52 or 53, wherein the probe has a 5′ flap.
 63. The method of claim52 or 53, wherein the RNA polymerase polymerizes nucleotidescomplementary to a length of the target sufficient to form a cleavagestructure so that the polymerized complementary RNA is adjacent at its3′ end to the probe.